WO2020056370A1 - Arnm modifié pour le traitement de troubles de la cholestase intrahépatique familiale progressive - Google Patents

Arnm modifié pour le traitement de troubles de la cholestase intrahépatique familiale progressive Download PDF

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WO2020056370A1
WO2020056370A1 PCT/US2019/051172 US2019051172W WO2020056370A1 WO 2020056370 A1 WO2020056370 A1 WO 2020056370A1 US 2019051172 W US2019051172 W US 2019051172W WO 2020056370 A1 WO2020056370 A1 WO 2020056370A1
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abcb4
mir
polynucleotide
sequence
utr
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PCT/US2019/051172
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WO2020056370A9 (fr
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Paolo Martini
Vladimir PRESNYAK
Jingsong Cao
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Modernatx, Inc.
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Priority to JP2021514132A priority Critical patent/JP2022500443A/ja
Priority to AU2019338557A priority patent/AU2019338557A1/en
Priority to CA3111836A priority patent/CA3111836A1/fr
Priority to EP19861226.9A priority patent/EP3863645A4/fr
Priority to US17/276,112 priority patent/US20220054653A1/en
Publication of WO2020056370A1 publication Critical patent/WO2020056370A1/fr
Publication of WO2020056370A9 publication Critical patent/WO2020056370A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)

Definitions

  • PFIC Progressive familial intrahepatic cholestasis
  • Partial external biliary diversion is a surgical approach that diverts bile from the gallbladder externally into an ileostomy bag, which can also be used to alleviate symptoms of PFICs.
  • PFICs often lead to liver transplant when liver dysfunction becomes severe. The disease is typically progressive, leading to fulminant liver failure and death in childhood, in the absence of liver transplantation.
  • PFIC type 1 (PFIC-l) is caused by mutations in the gene encoding ATPase, aminophospholipid transporter, class I, type 8B, member 1 (ATP8B1), which is also known as ATPase phospholipid transporter 8B1, BRIC1, FIC1, PFIC, and PFIC1.
  • ATP8B1 is a P-type ATPase protein that is responsible for phospholipid translocation across membranes.
  • PFIC type 2 (PFIC -2) is caused by a variety of mutations in ABCB11, which encodes the bile salt export pump (BSEP) protein.
  • PFIC type 3 (PFIC-3) is caused by a variety of mutations in ATP binding cassette subfamily B member 4 (ABCB4), which encodes a floppase. There is currently no effective treatment for the underlying genetic defect that leads to PFICs.
  • composition comprising a modified polynucleotide having an open reading frame (ORF) encoding ATP binding cassette subfamily B member 4 (ABCB4) formulated in a lipid nanoparticle (LNP) carrier.
  • ORF open reading frame
  • ABCB4 polynucleotide comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof.
  • the modified polynucleotide comprises at least one modified nucleoside.
  • the at least one modified nucleoside is selected from the group consisting of: pseudouridine, 1 -methyl-pseudouridine, 5- methylcytidine, 5-methyluridine, 2'-0-methyluridine, 2-thiouridine, 5-methoxyuridine and N6-methyladenosine.
  • the at least one modified nucleoside is a 5- methoxyuridine.
  • at least 30% of the uridine residues are 5- methoxyuridines.
  • the modified polynucleotide comprises a poly-A region, a Kozak sequence, a 3’ untranslated region, a 5’ untranslated region, an miRNA binding site, or any combination thereof.
  • the miRNA binding site is a miR-l42 binding site.
  • the uracil or thymine content of the ORF relative to the theoretical minimum uracil or thymine content of a nucleotide sequence encoding the ABCB4 polypeptide (%UTM or %TTM), is between about 100% and about 150%.
  • the ORF further comprises at least one low-frequency codon.
  • the ORF is at least 92% identical to ABCB4-C013, ABCB4-C022, or ABCB4- C09, or at least 91% identical to ABCB4-C01, ABCB4-C02, ABCB4-C03, ABCB4-C04, ABCB4-C05, ABCB4-C06, ABCB4-CO10, ABCB4-C011, ABCB4-C012, ABCB4-C015, ABCB4-C016, ABCB4-C017, ABCB4-CO20, ABCB4-C021, ABCB4-C023, ABCB4- C024, ABCB4-C025, or ABCB4-C026, or at least 90% identical to ABCB4-C07, ABCB4- C08, ABCB4-C014, ABCB4-C018, or ABCB4-C019.
  • the LNP comprises an ionizable amino lipid.
  • the ionizable amino lipid is compound 1.
  • a polynucleotide comprising an ORF, wherein the ORF is at least 92% identical to ABCB4-C013, ABCB4-C022, or ABCB4-C09, wherein the ORF is at least 91% identical to ABCB4-C01, ABCB4-C02, ABCB4-C03, ABCB4- C04, ABCB4-C05, ABCB4-C06, ABCB4-CO10, ABCB4-C011, ABCB4-C012, ABCB4- C015, ABCB4-C016, ABCB4-C017, ABCB4-CO20, ABCB4-C021, ABCB4-C023, ABCB4-C024, ABCB4-C025, or ABCB4-C026, or wherein the ORF is at least 90% identical to ABCB4-C07, ABCB4-C08, ABCB4-C014, ABCB4-C018, or ABCB4-C019.
  • the ABCB4 polypeptide comprises an amino acid sequence at least about 95% identical to (a) the polypeptide sequence of wild type ABCB4, isoform 1, (b) the polypeptide sequence of wild type ABCB4, isoform 2, or (c) the polypeptide sequence of wild type ABCB4, isoform 3, wherein the ABCB4 polypeptide has phosphatidylcholine translocation activity.
  • the ABCB4 polypeptide is a variant, derivative, or mutant having phosphatidylcholine translocation activity.
  • the polynucleotide encodes an ABCB4 polypeptide fused to one or more heterologous polypeptides.
  • the one or more heterologous polypeptides increase a pharmacokinetic property of the ABCB4 polypeptide.
  • the polynucleotide has: a longer plasma half-life, increased expression of an ABCB4 polypeptide encoded by the ORF, a lower frequency of arrested translation resulting in an expression fragment, greater structural stability, or any
  • the disclosure provides a method of producing a polynucleotide having an open reading frame (ORF) encoding ATP binding cassette subfamily B member 4 (ABCB4), comprising modifying an ORF encoding an ABCB4 polypeptide by performing at least one synonymous substitution.
  • ORF open reading frame
  • ABCB4 ATP binding cassette subfamily B member 4
  • at least 90% of uridine residues are replaced with 5-methoxyuridine.
  • the disclosure provides a method of treating or preventing progressive familial intrahepatic choleostasis type 3 (PFIC3) in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a composition comprising a modified mRNA molecule encoding an ABCB4 polypeptide.
  • the modified mRNA molecules comprises at least one modified nucleoside.
  • the at least one modified nucleoside is selected from the group consisting of: pseudouridine, 1 -methyl-pseudouridine, 5-methylcytidine, 5-methyluridine, 2'-0- methyluridine, 2-thiouridine, 5-methoxyuridine and N6-methyladenosine.
  • At least one modified nucleoside is a 5-methoxyuridine.
  • the modified mRNA molecule is formulated in a cationic lipid nanoparticle.
  • the disclosure provides a method of treating PFIC2 or BRIC2 in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a composition comprising a modified mRNA encoding a BSEP polypeptide.
  • the modified mRNA encoding a BSEP polypeptide comprises an open reading frame encoding ABCB11.
  • the disclosure provides a method of treating PFIC1 in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a composition comprising a modified mRNA encoding ATP8B1.
  • Fig. 1 depicts two Western blots confirming the expression of ABCB4 proteins in HEK293 cells transfected with ABCB4 modified RNA (modRNA) constructs.
  • the blots confirm ectopic expression of the human or mouse ABCB4 mRNA in transfected mammalian cells.
  • Anti-ABCB4 C-219 recognizes both hABCB4 and mABCB4, while anti-ABCB4 (R3P-26) is hABCB4-specific.
  • Fig. 2 is a graph showing the level of bile phosphatidylcholine in mdr2 knockout mice 24 hours after treatment with modRNAs.
  • Fig. 3 is a capillary electrophoresis (CE)-based simple Western image and a graph showing the relative ABCB4 expression resulting from HEK293 cells transfected with modRNAs.
  • the modRNAs in lanes 5-18 were codon-optimized. Earlier iterations of modRNAs (no codon-optimization) are shown in lanes 2-4.
  • Lane 1 is eGFP. Lanes 19 and 20 are mock transfected.
  • Fig. 4 is a graph showing the phosphatidylcholine transporting activity of different modRNAs. Bars 1-3 show modRNAs that were not codon-optimized. Bars 4-12 and modRNA5, modRNA6, modRNA7, modRNA8, modRNA9 show codon-optimized modRNAs. The arrows indicate modRNAs (modRNA5, modRNA6, modRNA7, modRNA8, and modRNA9) selected for further analysis.
  • Fig. 5 is two graphs showing the in vivo phosphatidylcholine transporting activity of different modRNAs in mdr2 knockout mice before codon optimization (left graph; bar 1) and after codon optimization (right graph; modRNA5-modRNA9).
  • Fig. 6 shows the hepatic expression of hABCB4 protein in mdr2 knockout mice injected with modRNA7 mRNA at 1 mg/kg.
  • Fig. 7 shows an immunofluorescent study.
  • hABCB4 modRNA was found to result in expression of the ABCB4 protein at the canalicular domain of hepatocytes.
  • the tight junctional associated protein zonula occuludens-l (ZO-l) was used to visualize the border of the bile canalicular structures.
  • Fig. 8 is a graph showing the total bile acid levels in serum 24 hours after injection of eGFP or ABCB4 modRNA in Mdr2 knockout mice. The wild-type mice were untreated.
  • Fig. 9 includes two graphs relating to a kinetic analysis of bile phosphatidylcholine output in Mdr2 knockout mice after a single injection of hABCB4 modRNA formulated in a compound 3 lipid nanoparticle.
  • Fig. 10 is a graph showing a kinetic study of the total bile acids in serum.
  • Fig. 11 shows two graphs depicting the phosphatidylcholine output in bile using modRNA formulated in two different nanocarriers.
  • Fig. 12 is a series of graphs showing the efficacy of the second formulation of modRNA in compound 1 lipid nanoparticles.
  • Fig. 13 is a graph showing the bile phosphatidylcholine level as a percentage of wild- type in mice after the first injection and after the fifth injection.
  • Fig. 14 is a graph showing the total bile acids in serum. There was a significant reduction in the mice that received hABCB4 modRNA.
  • Figs. 15A-15C show improvements associated with hABCB4 modRNA treatment.
  • Fig. 15A shows subject body weight throughout the study.
  • Fig. 15B shows liver enzyme measurements and
  • Fig. 15C shows liver weight, portal pressure, and the phosphatidylcholine level in bile as a percentage of wild-type.
  • Fig. 16 shows fibrotic progression in the Mdr2-/- mice treated with hABCB4 mRNA.
  • the figure depicts connective tissue staining and a graphical representation of the fibrotic progression in mdr2 mice treated with hABCB4 modRNA. Sirius Red was used for staining purposes. Collagen content was also examined.
  • Fig. 17 is a series of graphs showing real-time PCR of various markers in liver fibrosis and inflammation.
  • Figs. 18A-18C show an evaluation of liver fibrosis using immunohistochemical staining. HE staining was also carried out to see the histological change of livers after different treatments (bottom panel of Fig. 18C).
  • Fig. 19 is a series of images illustrating that multiple injections of hABCB4 modRNA largely improve the expression of hABCB4 protein in the canalicular domain.
  • Fig. 20 is a schematic representation of the domain structure of isoform 1 of ABCB4.
  • Fig. 21 is a schematic representation of the domain structure of isoform 2 of ABCB4.
  • Fig. 22 is a schematic representation of the domain structure of isoform 3 of ABCB4.
  • Fig. 23 is a plot showing the half-life of human BSEP. Protein levels were measured using capillary electrophoresis with anti-ABCBl 1 for detection. Expression was measured in HepaRG cells modified by knocking out endogenous BSEP expression.
  • Fig. 24 shows a schematic of an in vitro assay for BSEP activity and representative results. Titrated TCA were measured in the presence of a control (GFP) and expressed BSEP (mABCBl 1) in the middle panel. The lower panel shows reduced bile acids at least four days post-transfection.
  • Fig. 25 shows immunostaining of cells transfected with GFP (control) or hABCBl 1 modRNA in HepaRG cells (Fig. 23).
  • Fig. 26 is a plot showing delivery of modRNA to the liver of wild-type mice.
  • Fig. 27 depicts immunostained images showing protein expression in mice on a regular diet and on a model-inducing cholic acid diet. Protein is associated with membrane and expression is increased in the cholic acid-fed mice.
  • nucleic acid molecules including modified nucleic acid molecules, and methods of using the same, for example, to treat progressive familial intrahepatic cholestasis disorders.
  • the nucleic acid molecules including RNAs such as mRNAs, contain, for example, one or more modifications that improve properties of the molecule.
  • Such improvements include, but are not limited to, increased stability and/or clearance in tissues, improved receptor uptake and/or kinetics, improved cellular access by the compositions, improved engagement with translational machinery, improved mRNA half- life, increased translation efficiency, improved immune evasion, improved protein production capacity, improved secretion efficiency, improved accessibility to circulation, improved protein half-life and/or modulation of a cell’s status, improved function and/or improved activity.
  • the present disclosure provides compositions of nucleic acids relating to biliary epithelial transporters.
  • the present disclosure relates to nucleic acids capable of regulating the biliary secretion of phospholipids, including phosphatidylcholine, e.g., those encoded by ABCB4 or a biologically active fragment thereof, in a target cell.
  • the present disclosure relates to nucleic acids capable of regulating protein expression of a bile salt export pump (BSEP), e.g., that encoded by ABCB11, or a biologically active fragment thereof in a target cell.
  • BSEP bile salt export pump
  • the present disclosure provides nucleic acids related to the catalyzation of ATP hydrolysis coupled to the transport of aminophospholipids (e.g, phosphatidyl serine and phosphatidylethanolamine), e.g, those encoded by ATP8B1 or a biologically active fragment thereof, in a target cell.
  • aminophospholipids e.g, phosphatidyl serine and phosphatidylethanolamine
  • compositions provided herein are useful for treating diseases or disorder associated with a deficiency of biliary epithelial transporters, such as progressive familial intrahepatic cholestasis (PFIC).
  • PFIC refers to a group of familial cholestatic conditions caused by defects in biliary epithelial transporters. Srivastava, J. Clin. Exp. Hepatol. 4(1): 25- 36 (2014); Morotti et ak, Seminars in Liver Disease 31(1): 3-10 (2011); and Stapelbroek et al., J. Hepatol. 52: 258-271 (2010).
  • PFIC has been classified into three types (types 1, 2 and 3) based on the genetic defect involved in the transporter. PFIC3 is caused by mutation of ABCB4.
  • PFIC PFIC
  • infancy Signs and symptoms of PFIC typically begin in infancy and are related to bile acid buildup and liver disease. Specifically, affected individuals experience severe itching, yellowing of the skin and whites of the eyes (jaundice), failure to gain weight and grow at the expected rate, high blood pressure in the vein that supplies blood to the liver (portal hypertension), and an enlarged liver and spleen (hepatosplenomegaly).
  • PFIC3 most people with PFIC3 have signs and symptoms related to liver disease only. Also, the signs and symptoms of PFIC3 usually do not appear until later in infancy or early childhood. Liver failure can occur in childhood or adulthood in people with PFIC3.
  • compositions of nucleic acids capable of regulating protein expression of a bile salt export pump e.g., that is encoded by ABCB11, or a biologically active fragment thereof in a target cell.
  • BSEP bile salt export pump
  • the compositions provided herein are useful for treating diseases or disorder associated with a deficiency of BSEP activity, such as, for example, benign recurrent intrahepatic cholestasis 2 (BRIC2) and progressive familial intrahepatic cholestasis (PFIC), e.g., PFIC2.
  • BRIC2 benign recurrent intrahepatic cholestasis 2
  • PFIC progressive familial intrahepatic cholestasis
  • Nucleic acids include, for example, polynucleotides, which further include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs; Yu, H. et al., Nat. Chem.,
  • glycol nucleic acids for reviews see LTeda, N. et al., J. Het. Chem., 8:827 9, 1971; Zhang, L. et al., J. Am. Chem. Soc., 127:4174 5, 2005
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • compositions provided herein are useful for treating health issues associated with mutations in ATP8B1, including, but not limited to, are progressive familial intrahepatic cholestasis type 1 (PFIC-l), benign recurrent intrahepatic cholestasis type 1 (BRIC1), and intrahepatic cholestasis of pregnancy type 1 (ICP1).
  • PFIC-l progressive familial intrahepatic cholestasis type 1
  • BRIC1 benign recurrent intrahepatic cholestasis type 1
  • ICP1 intrahepatic cholestasis of pregnancy type 1
  • PFIC-l is a disorder characterized by early onset of cholestasis that progresses to hepatic fibrosis, cirrhosis, and end-stage liver disease before adulthood.
  • BRIC1 is a disorder characterized by
  • ICP1 is a liver disorder of pregnancy that presents during the second or third trimester with intense pruritus, which becomes more severe with advancing gestation, and cholestasis. Id. ICP1 causes fetal distress, spontaneous premature delivery and intrauterine death. ICP1 patients have spontaneous and progressive
  • nucleic acid-based therapeutics e.g., mRNA therapeutics
  • mRNA therapeutics e.g., mRNA therapeutics
  • TLRs toll-like receptors
  • ssRNA single-stranded RNA
  • RAG-I retinoic acid-inducible gene I
  • Immune recognition of foreign mRNAs can result in unwanted cytokine effects including interleukin- 1b (IL- 1 b) production, tumor necrosis factor-a (TNF-a) distribution, and a strong type I interferon (type I IFN) response.
  • the instant invention features the incorporation of different modified nucleotides within therapeutic mRNAs to minimize the immune activation and optimize the translation efficiency of mRNA to protein.
  • Particular aspects of the invention feature a combination of nucleotide modification to reduce the innate immune response and sequence optimization, in particular, within the open reading frame (ORF) of therapeutic mRNAs encoding ABCB4, ABCB11, and/or ATP8B1 to enhance protein expression.
  • ORF open reading frame
  • the mRNA therapeutic technology of the instant invention also features delivery of mRNA encoding ABCB4, ABCB11, and/or ATP8B1 via a lipid nanoparticle (LNP) delivery system.
  • LNPs lipid nanoparticles
  • LNPs are an ideal platform for the safe and effective delivery of mRNAs to target cells.
  • LNPs have the unique ability to deliver nucleic acids by a mechanism involving cellular uptake, intracellular transport and endosomal release or endosomal escape.
  • the instant invention features ionizable amino lipid-based LNPs which have improved properties when administered in vivo.
  • the ionizable amino lipid-based LNPs of the invention have improved properties, for example, cellular uptake, intracellular transport and/or endosomal release or endosomal escape.
  • LNPs administered by systemic route e.g., intravenous (IV)
  • mRNA engineering and/or efficient delivery by LNPs can result in increased levels and or enhanced duration of protein being expressed following a first dose of administration, which in turn, can lengthen the time between first dose and subsequent dosing.
  • accelerated blood clearance (ABC) phenomenon is, at least in part, transient in nature, with the immune responses underlying ABC resolving after sufficient time following systemic administration.
  • increasing the duration of protein expression and/or activity following systemic delivery of an mRNA therapeutic of the invention in one aspect combats the ABC phenomenon.
  • LNPs can be engineered to avoid immune sensing and/or recognition and can thus further avoid ABC upon subsequent or repeat dosing.
  • Exemplary aspect of the invention feature novel LNPs which have been engineered to have reduced ABC.
  • kits and devices for the design, preparation, manufacture and formulation of such nucleic acids are also included in the instant disclosure.
  • ABSB4 ATP Binding Cassete Subfamily B Member 4
  • ABCB4 ATP binding cassette subfamily B member 4
  • MDR3, MDR2/3, MDR/TAP, P-glycoprotein 3, or PGY3 ATP binding cassette subfamily B member 4
  • PGY3 ATP binding cassette subfamily B member 4
  • ABCB4 has three isoforms: isoform A (isoform 2) contains 1,279 amino acid residues (GenBank Accession Nos. NP_000434. l and
  • isoform B (isoform 1) contains 1,286 amino acid residues (GenBank
  • isoform C contains 1,232 amino acid residues (GenBank Accession Nos. NP_06l338.l and NM_0l8850.2).
  • Isoform A lacks amino acid residues 1094-1100 of isoform B.
  • Isoform C lacks amino acid residues 929- 975 and 1094-1100 of isoform B.
  • Figs. 21-23 show schematic representations of each isoform.
  • ABCB4 is responsible for biliary secretion of phospholipids, predominantly phosphatidylcholine. Defective phosphatidylcholine translocation leads to a lack of phosphatidylcholine in bile fluid. Phosphatidylcholine normally chaperones bile acids, preventing damage to the biliary epithelium. As such, PFIC3/ABCB4 deficiency can result in injury to the biliary epithelium and bile canaliculi, cholestasis, high serum g- glutamyltranspeptidase (GGT) levels, and cholesterol gallstone disease. ATP Binding Cassete Subfamily B Member 11 (ABCB11)
  • ATP binding cassette, subfamily B, member 11 encodes a Bile Salt Export Pump (BSEP, aka sPgp (sister of P-glycoprotein)).
  • the deduced protein has a predicted topology similar to that of other members of the multidrug resistant (MDR) family, with two putative transmembrane domains, each with six spans, and two nucleotide binding folds containing highly conserved ATP binding cassettes (ABC).
  • Northern blot analysis indicates the ABCB11 gene produces a 5.5 kb mRNA transcript in liver.
  • Ten different alleles of the ABCB11 gene have been identified in PFIC2 patients. Four alleles cause premature termination of the protein; the remaining alleles were missense changes. Five of the missense alleles were found in consanguineous families and the affected individuals were all homozygous for the mutation.
  • An exemplary protein sequence for BSEP encoded by human ABCB11 is published as NCBI reference no. NP_003733.2.
  • ATPase Aminophospholipid Transporter, Class I, Type 8B, Member 1 (ATP8B1)
  • ATPase aminophospholipid transporter, class I, type 8B, member 1 (ATP8B1; EC 3.6.3.1) is a member of the P-type cation transport ATPase family and belongs to the subfamily of aminophospholipid-transporting ATPases.
  • ATP8B1 serves as the catalytic component of a P4-ATPase flippase complex. Paulusma, C.C. et al ., Hepatology 47:268-278 (2008).
  • the P4-ATPase flippase complex catalyzes the hydrolysis of ATP coupled to the transport of aminophospholipids, such as phosphatidylserine and phosphatidylethanolamine, from the outer to the inner leaflet of various membranes, thus ensuring the maintenance of asymmetric distribution of phospholipids. Id.
  • Phospholipid translocation is implicated in vesicle formation and in uptake of lipid signaling molecules. It is also required for the preservation of cochlear hair cells in the inner ear. See , e.g., Munoz -Martinez, F. et al., Biochem. Pharmacol. 80:793-800 (2010) and Verhultst, P.M. et al., Hepatology 51:2049-2060 (2010).
  • Phospholipid translocation can further play a role in asymmetric distribution of phospholipids in the canicular membrane, transport of bile acids into the canaliculus, uptake of bile acids from intestinal contents into intestinal mucosa, protecting hepatocytes from bile salts by establishing integrity of the canalicular membrane in cooperation with ABCB4, microvillus formation in polarized epithelial cells, and as a cardiolipin transporter during inflammatory injury. Id.
  • the coding sequence (CDS) for wild-type ATP8B1 canonical mRNA sequence is described at the NCBI Reference Sequence database (RefSeq) under accession number NM 005603.4 (Homo sapiens ATPase phospholipid transporting 8B1 (ATP8B1), mRNA).
  • the wild-type ATP8B1 canonical protein sequence is described at the RefSeq database under accession number NP 005594.1 (phospholipid-transporting ATPase IC [Homo sapiens]).
  • the ATP8B1 protein is 1251 amino acids long. It is noted that the specific nucleic acid sequences encoding the reference protein sequence in the RefSeq sequences are the coding sequence (CDS) as indicated in the respective RefSeq database entry.
  • the disclosure provides a polynucleotide (e.g., a RNA, e.g., a mRNA) comprising a nucleotide sequence (e.g., an open reading frame (ORF)) encoding an ABCB4, ABCB11, or ATP8B1 polypeptide.
  • the ABCB4 polypeptide of the invention is a wild type full length human ABCB4 isoform 1, 2, or 3 protein.
  • the ABCB11 polypeptide of the invention is a wild type full length human ABCB11 protein.
  • the ATP8B1 polypeptide of the invention is a wild type full length human ATP8B1 protein.
  • the ABCB4 polypeptide, ABCB11 polypeptide, or ATP8B1 polypeptide of the invention is a variant, a peptide or a polypeptide containing a substitution, and insertion and/or an addition, a deletion and/or a covalent modification with respect to a wild-type ABCB4 isoform 1, 2, or 3 sequence, wild- type ABCB11 sequence, or wild-type ATP8B1 sequence.
  • sequence tags or amino acids can be added to the sequences encoded by the polynucleotides of the invention (e.g., at the N-terminal or C-terminal ends), e.g., for localization.
  • amino acid residues located at the carboxy, amino terminal, or internal regions of a polypeptide of the invention can optionally be deleted providing for fragments.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide encodes a substitutional variant of a human ABCB4 isoform 1, 2, or 3 sequence, a human ABCB11 sequence, or a human
  • the substitutional variant can comprise one or more conservative amino acids substitutions.
  • the variant is an insertional variant. In other embodiments, the variant is a deletional variant.
  • ABCB4, ABCB11, and ATP8B1 protein fragments, functional protein domains, variants, and homologous proteins (orthologs) are also within the scope of the ABCB4, ABCB11, and ATP8B1 polypeptides of the disclosure.
  • polypeptides encoded by the polynucleotides of the invention are shown in SEQ ID NO: 1 (ABCB4, isoform 2), SEQ ID NO: 3 (ABCB4, isoform 1), SEQ ID NO: 5 (ABCB4, isoform 3), SEQ ID NO: 7 (ABCB11), and SEQ ID NO: 9 (ATP8B1).
  • Certain compositions and methods presented in this disclosure refer to the protein or polynucleotide sequences of wild type human ABCB4 isoform 1, 2, or 3. Such disclosures are equally applicable to other isoforms of ABCB4.
  • the instant invention features mRNAs for use in treating or preventing progressive familial intrahepatic cholestasis (PFIC).
  • the mRNAs featured for use in the invention are administered to subjects and encode human ABCB4, ABCB11, and/or ATP8B1 protein in vivo.
  • the invention relates to polynucleotides, e.g., mRNA, comprising an open reading frame of linked nucleosides encoding human ABCB4 (SEQ ID NO: 1), ABCB11 (SEQ ID NO: 7), or ATP8B1 (SEQ ID NO: 9), isoforms thereof, functional fragments thereof, and fusion proteins comprising ABCB4, ABCB11, or ATP8B1.
  • the open reading frame is sequence-optimized.
  • the invention provides sequence-optimized polynucleotides comprising nucleotides encoding the polypeptide sequence of human ABCB4, human ABCB11, or human ATP8B1, or sequence having high sequence identity with those sequence optimized polynucleotides.
  • the invention provides polynucleotides (e.g., a RNA such as an mRNA) that comprise a nucleotide sequence (e.g., an ORF) encoding one or more ABCB4, ABCB11, and/or ATP8B1 polypeptides.
  • a nucleotide sequence e.g., an ORF
  • ABCB11, or ATP8B1 polypeptide of the invention can be selected from: a full length ABCB4, ABCB11, or ATP8B1 polypeptide (e.g., having the same or essentially the same length as wild-type ABCB4 isoform 1, 2, or 3, wild-type ABCB11, or ATP8B1); a functional fragment of ABCB4, ABCB11, or ATP8B1 described herein (e.g., a truncated (e.g., deletion of carboxy, amino terminal, or internal regions) sequence shorter than wild-type ABCB4, ABCB11, or ATP8B1; but still allowing for ABCB4, ABCB11, or ATP8B1 activity); a variant thereof (e.g., full length or truncated ABCB4, ABCB11, or ATP8B1 proteins in which one or more amino acids have been replaced, e.g., variants that retain all or most of the ABCB4, ABCB11, or ATP8B1 activity of the polypeptide with
  • the encoded ABCB4 polypeptide is a mammalian ABCB4 polypeptide, such as a human ABCB4 polypeptide, a functional fragment or a variant thereof.
  • the encoded ABCB11 polypeptide is a mammalian ABCB11 polypeptide, such as a human ABCB11 polypeptide, a functional fragment or a variant thereof.
  • the encoded ATP8B1 polypeptide is a mammalian ATP8B1 polypeptide, such as a human ATP8B1 polypeptide, a functional fragment or a variant thereof.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention increases ABCB4, ABCB11, or ATP8B1 protein expression levels and/or detectable bile transport levels in cells when introduced in those cells, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, compared to ABCB4, ABCB11, or
  • the polynucleotide is introduced to the cells in vitro. In some embodiments, the polynucleotide is introduced to the cells in vivo.
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the invention comprise a nucleotide sequence (e.g., an ORF) that encodes a wild-type human ABCB4, e.g., wild-type isoform 2 of human ABCB4 (SEQ ID NO: 1).
  • a nucleotide sequence e.g., an ORF
  • a wild-type human ABCB4 e.g., wild-type isoform 2 of human ABCB4 (SEQ ID NO: 1).
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence (e.g., an ORF) that encodes a wild-type human ABCB11 (SEQ ID NO: 7).
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence (e.g., an ORF) that encodes a wild-type human ATP8B1, e.g., wild-type human ATP8B1 (SEQ ID NO: 9).
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a codon optimized nucleic acid sequence, wherein the open reading frame (ORF) of the codon optimized nucleic acid sequence is derived from a wild-type ABCB4, ABCB11, or ATP8B1 sequence (e.g., wild-type human ABCB4, a wild-type human ABCB11, or a wild-type human ATP8B1).
  • ORF open reading frame
  • the corresponding wild type sequence is the native human ABCB4, ABCB11, or ATP8B1.
  • the corresponding wild type sequence is the corresponding fragment from human ABCB4, ABCB11, or ATP8B1.
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence encoding ABCB4 isoform 2 having the full length sequence of human ABCB4 isoform 2 (i.e., including the initiator methionine).
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence encoding ABCB4 isoform 1 having the full length sequence of human ABCB4 isoform 1 (i.e., including the initiator methionine).
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence encoding ABCB4 isoform 3 having the full length sequence of human ABCB4 isoform 3 (i.e., including the initiator methionine).
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the invention comprise a nucleotide sequence encoding ABCB11 isoform 1 having the full length sequence of human ABCB11.
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence encoding ATP8B1 having the full length sequence of human ATP8B1 (i.e., including the initiator methionine).
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprising a nucleotide sequence encoding ABCB4, ABCB11, or ATP8B1 having the full length or mature sequence of human ABCB4,
  • ABCB11, or ATP8B1 is sequence optimized.
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the invention comprise a nucleotide sequence (e.g., an ORF) encoding a mutant ABCB4,
  • the polynucleotides of the invention comprise an ORF encoding an ABCB4, ABCB11, or ATP8B1 polypeptide that comprises at least one point mutation in the ABCB4, ABCB11, or ATP8B1 amino acid sequence and retains bile transport activity.
  • the mutant ABCB4, ABCB11, or ATP8B1 polypeptide causes a bile transport activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the bile transport activity resulting from the corresponding wild-type ABCB4, ABCB11, or ATP8B1 (i.e., the same ABCB4, ABCB11, or ATP8B1 isoform but without the mutation(s)).
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprising an ORF encoding a mutant ABCB4, ABCB11, or ATP8B1 polypeptide is sequence optimized.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) that encodes an ABCB4, ABCB11, or ATP8B1 polypeptide with mutations that do not alter bile transport activity.
  • a mutant ABCB4, ABCB11, or ATP8B1 polypeptides can be referred to as function-neutral.
  • the polynucleotide comprises an ORF that encodes a mutant ABCB4,
  • the mutant ABCB4, ABCB11, or ATP8B1 polypeptide has higher bile transport activity than the corresponding wild-type ABCB4, ABCB11, or
  • the mutant ABCB4, ABCB11, or ATP8B1 polypeptide causes bile transport activity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% higher than the activity of the corresponding wild-type ABCB4, ABCB11, or ATP8B1 (i.e., the same ABCB4, ABCB11, or ATP8B1 isoform but without the mutation(s)).
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the invention comprise a nucleotide sequence (e.g., an ORF) encoding a functional ABCB4, ABCB11, or ATP8B1 fragment, e.g., where one or more fragments correspond to a polypeptide subsequence of a wild type ABCB4, ABCB11, or ATP8B1 polypeptide and retain bile transport activity.
  • the ABCB4, ABCB11, or ATP8B1 fragment causes bile transport activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the bile transport activity of the corresponding full length ABCB4, ABCB11, or ATP8B1.
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the invention comprising an ORF encoding a functional ABCB4,
  • ABCB11, or ATP8B1 fragment are sequence optimized.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB4, ABCB11, or ATP8B1 fragment that causes higher bile transport activity than the corresponding full length ABCB4, ABCB11, or ATP8B1.
  • a nucleotide sequence e.g., an ORF
  • the ABCB4, ABCB11, or ATP8B1 fragment causes a bile transport activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% higher than the bile transport activity of the
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB4 fragment that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% shorter than wild-type ABCB4 isoform 1, 2, or 3.
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB11 fragment that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% shorter than wild-type ABCB11.
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ATP8B1 fragment that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB4 polypeptide (e.g., the sequence depicted in SEQ ID NO: 1, functional fragment, or variant thereof), wherein the nucleotide sequence has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB11 polypeptide (e.g., the sequence depicted in SEQ ID NO:7, functional fragment, or variant thereof), wherein the nucleotide sequence has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ATP8B1 polypeptide (e.g., the sequence depicted in SEQ ID NO:9, functional fragment, or variant thereof), wherein the nucleotide sequence has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises an ORF encoding an ABCB4 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the polynucleotide comprises a nucleic acid sequence having 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 70% to 95%, 80% to 95%, 70% to 85%, 75% to 90%, 80% to 95%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, sequence identity to a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 68-92, 118, 120, 122, and 124.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises an ORF encoding an ABCB11 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the polynucleotide comprises a nucleic acid sequence having 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 70% to 95%, 80% to 95%, 70% to 85%, 75% to 90%, 80% to 95%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, sequence identity to a sequence selected from the group consisting of SEQ ID NO: 8, 126, 128, 130, 132, 134, 136, 138, 140, and 142.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises an ORF encoding an ATP8B1 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the polynucleotide comprises a nucleic acid sequence having 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 70% to 95%, 80% to 95%, 70% to 85%, 75% to 90%, 80% to 95%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, sequence identity to a sequence selected from the group consisting of SEQ ID NO: 10, 93-117.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB4 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the nucleotide sequence is between 70% and 90% identical; between 75% and 85% identical; between 76% and 84% identical; between 77% and 83% identical, between 77% and 82% identical, or between 78% and 81% identical to a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 68-92, 118, 120, 122, and 124.
  • a nucleotide sequence e.g., an ORF
  • ABCB4 polypeptide e.g., the wild-type sequence, functional fragment, or variant thereof
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • the polynucleotide of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB11 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the nucleotide sequence is between 70% and 90% identical;
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ATP8B1 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the nucleotide sequence is between 70% and 90% identical; between 75% and 85% identical; between 76% and 84% identical; between 77% and 83% identical, between 77% and 82% identical, or between 78% and 81% identical to a sequence selected from the group consisting of SEQ ID NO: 10, 93-117.
  • a nucleotide sequence e.g., an ORF
  • an ATP8B1 polypeptide e.g., the wild-type sequence, functional fragment, or variant thereof
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises from about 900 to about 100,000 nucleotides (e.g., from 900 to 1,000, from 900 to 1,100, from 900 to 1,200, from 900 to 1,300, from 900 to 1,400, from 900 to
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB4, ABCB11, or ATP8B1 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the length of the nucleotide sequence (e.g., an ORF) is at least 500 nucleotides in length (e.g, at least or greater than about 500, 600, 700, 80, 900, 1,000, 1,050, 1,100, 1,187, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400,
  • a nucleotide sequence e.g., an ORF
  • the length of the nucleotide sequence e.g., an ORF
  • the length of the nucleotide sequence e.g.
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB4, ABCB11, or ATP8B1 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof) further comprises at least one nucleic acid sequence that is noncoding, e.g., a microRNA binding site.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention further comprises a 5'-UTR (e.g., selected from the sequences of SEQ ID NO: 12, , 172, 183, 184, 186-189, 191-194, 223-239, 287 and 288) and a 3'UTR (e.g., selected from the sequences of SEQ ID NO: 13, 154, 170-173, 176-185, 190-205, and 206-222).
  • a 5'-UTR e.g., selected from the sequences of SEQ ID NO: 12, , 172, 183, 184, 186-189, 191-194, 223-239, 287 and 28
  • a 3'UTR e.g., selected from the sequences of SEQ ID NO: 13, 154, 170-173, 176-185, 190-205, and 206-222.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a sequence selected from the group consisting of SEQ ID NO: 12 172, 183, 184, 186-189, 191- 194, 223-239, 287 and 288.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 5' terminal cap (e.g., CapO, Capl, ARCA, inosine, Nl-methyl- guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof) and a poly-A-tail region (e.g., about 100 nucleotides in length).
  • a 5' terminal cap e.g., CapO, Capl, ARCA, inosine, Nl-methyl- guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 3' UTR comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, 206-222, or any combination thereof.
  • the mRNA comprises a 3' UTR comprising a nucleic acid sequence of SEQ ID NO: 13.
  • the mRNA comprises a poly-A tail.
  • the poly-A tail is 50-150, 75-150, 85-150, 90-150, 90-120, 90-130, or 90-150 nucleotides in length.
  • the poly-A tail is 100 nucleotides in length.
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB4, ABCB11, or ATP8B1 polypeptide is single stranded or double stranded.
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide of the invention comprising a nucleotide sequence (e.g., an ORF) encoding an ABCB4, ABCB11, or ATP8B1 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof) is DNA or RNA.
  • the polynucleotide of the invention is RNA.
  • the polynucleotide of the invention is, or functions as, an mRNA.
  • the mRNA comprises a nucleotide sequence (e.g., an ORF) that encodes at least one ABCB4, ABCB11, or ATP8B1 polypeptide, and is capable of being translated to produce the encoded ABCB4, ABCB11, or ATP8B1 polypeptide in vitro, in vivo, in situ or ex vivo.
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a sequence-optimized nucleotide sequence (e.g., an ORF) encoding an ABCB4, ABCB11, or ATP8B1 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof, see e.g., SEQ ID NO: 1, 3, 5, 7, 9), wherein the polynucleotide comprises at least one chemically modified nucleobase, e.g., Nl-methylpseudouracil or 5- methoxyuracil. In certain embodiments, all uracils in the polynucleotide are
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miR-l42 and/or a miRNA binding site that binds to miR-l26.
  • the polynucleotide (e.g., a RNA, e.g., a mRNA) disclosed herein is formulated with a delivery agent comprising, e.g., a compound having the Formula (I), e.g., any of Compounds 1-232, e.g., Compound II; a compound having the Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound VI; or a compound having the Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound I, or any combination thereof.
  • the delivery agent comprises Compound II, DSPC, Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio of about
  • the delivery agent comprises Compound VI, DSPC, Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio in the range of about 30 to about 60 mol% Compound II or VI (or related suitable amino lipid) (e.g., 30-40, 40-45, 45- 50, 50-55 or 55-60 mol% Compound II or VI (or related suitable amino lipid)), about 5 to about 20 mol% phospholipid (or related suitable phospholipid or“helper lipid”) (e.g., 5-10, 10-15, or 15-20 mol% phospholipid (or related suitable phospholipid or“helper lipid”)), about 20 to about 50 mol% cholesterol (or related sterol or“non-cationic” lipid) (e.g., about 20-30, 30-35, 35-40, 40-45, or 45-50 mol% cholesterol (or related sterol or“non-cationic” lipid)) and about 0.05 to about
  • An exemplary delivery agent can comprise mole ratios of, for example, 47.5: 10.5:39.0:3.0 or 50: 10:38.5: 1.5. In certain instances, an exemplary delivery agent can comprise mole ratios of, for example, 47.5: 10.5:39.0:3; 47.5: 10:39.5:3; 47.5: 11 :39.5:2; 47.5:10.5:39.5:2.5; 47.5: 11 :39:2.5;
  • the delivery agent comprises
  • the delivery agent comprises Compound II or VI, DSPC, Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio of about 50: 10:38.5: 1.5.
  • the polynucleotide of the disclosure is an mRNA that comprises a 5'-terminal cap (e.g., Cap 1), a 5'UTR (e.g., SEQ ID NO: 12), a ORF sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 68-118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, a 3'UTR (e.g., SEQ ID NO: 13), and a poly-A tail (e.g., about 100 nucleotides in length), wherein all uracils in the polynucleotide are
  • the delivery agent comprises Compound II or Compound VI as the ionizable lipid and PEG-DMG or Compound I as the PEG lipid.
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • One such feature that aids in protein trafficking is the signal sequence, or targeting sequence.
  • the peptides encoded by these signal sequences are known by a variety of names, including targeting peptides, transit peptides, and signal peptides.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) that encodes a signal peptide operably linked to a nucleotide sequence that encodes an ABCB4, ABCB11, or ATP8B1 polypeptide described herein.
  • a nucleotide sequence e.g., an ORF
  • a signal peptide operably linked to a nucleotide sequence that encodes an ABCB4, ABCB11, or ATP8B1 polypeptide described herein.
  • the "signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 30-210, e.g., about 45-80 or 15-60 nucleotides (e.g., about 20, 30, 40, 50, 60, or 70 amino acids) in length that, optionally, is incorporated at the 5' (or N-terminus) of the coding region or the polypeptide, respectively. Addition of these sequences results in trafficking the encoded polypeptide to a desired site, such as the endoplasmic reticulum or the mitochondria through one or more targeting pathways. Some signal peptides are cleaved from the protein, for example by a signal peptidase after the proteins are transported to the desired site.
  • the polynucleotide of the invention comprises a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, wherein the nucleotide sequence further comprises a 5' nucleic acid sequence encoding a heterologous signal peptide.
  • the polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • polynucleotides of the invention comprise a single ORF encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, a functional fragment, or a variant thereof.
  • the polynucleotide of the invention can comprise more than one ORF, for example, a first ORF encoding an ABCB4, ABCB11, or ATP8B1 polypeptide (a first polypeptide of interest), a functional fragment, or a variant thereof, and a second ORF expressing a second polypeptide of interest.
  • a first ORF encoding an ABCB4, ABCB11, or ATP8B1 polypeptide (a first polypeptide of interest), a functional fragment, or a variant thereof
  • a second ORF expressing a second polypeptide of interest.
  • two or more polypeptides of interest can be genetically fused, i.e., two or more polypeptides can be encoded by the same ORF.
  • the polynucleotide can comprise a nucleic acid sequence encoding a linker (e.g., a G 4 S (SEQ ID NO: 11) peptide linker or another linker known in the art) between two or more polypeptides of interest.
  • a polynucleotide of the invention can comprise two, three, four, or more ORFs, each expressing a polypeptide of interest.
  • a polynucleotide of the invention can comprise at least three ORFs: a first ORF encoding ABCB4, a second ORF encoding ABCB11, and a third ORF encoding ATP8B1.
  • the polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • a first nucleic acid sequence e.g., a first ORF
  • a second nucleic acid sequence e.g., a second ORF
  • the mRNAs of the disclosure encode more than one ABCB4, ABCB11, or ATP8B1 domain or a heterologous domain, referred to herein as multimer constructs.
  • the mRNA further encodes a linker located between each domain.
  • the linker can be, for example, a cleavable linker or protease-sensitive linker.
  • the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, ATP8B1A linker, and combinations thereof.
  • the linker is an F2A linker.
  • the linker is a GGGS (SEQ ID NO: 240) linker.
  • the multimer construct contains three domains with intervening linkers, having the structure: domain-linker-domain- linker-domain e.g., ABCB4, ABCB11, or ATP8B1 domain-linker-ABCB4, ABCB11, or ATP8B1 domain-linker- ABCB4, ABCB11, or ATP8B1 domain.
  • the cleavable linker is an F2A linker (e.g., having the amino acid sequence GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 242)).
  • the cleavable linker is a T2A linker (e.g., having the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:243)), a P2A linker (e.g, having the amino acid sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:244)) or an ATP8B1A linker (e.g, having the amino acid sequence GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:245)).
  • T2A linker e.g., having the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:243)
  • P2A linker e.g, having the amino acid sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:244)
  • an ATP8B1A linker e.g, having the amino acid sequence GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:245)
  • the skilled artisan will appreciate that other art
  • the self-cleaving peptide may be, but is not limited to, a 2A peptide.
  • 2A peptides are known and available in the art and may be used, including e.g, the foot and mouth disease virus (FMDV) 2A peptide, the equine rhinitis A virus 2A peptide, the Thosea asigna virus 2A peptide, and the porcine teschovirus-l 2A peptide.
  • FMDV foot and mouth disease virus
  • 2A peptides are used by several viruses to generate two proteins from one transcript by ribosome-skipping, such that a normal peptide bond is impaired at the 2A peptide sequence, resulting in two discontinuous proteins being produced from one translation event.
  • the 2A peptide may have the protein sequence of SEQ ID NO:244, fragments or variants thereof. In one embodiment, the 2A peptide cleaves between the last glycine and last proline.
  • the polynucleotides of the present invention may include a polynucleotide sequence encoding the 2A peptide having the protein sequence of fragments or variants of SEQ ID NO:244.
  • polynucleotide sequence encoding the 2A peptide is:
  • a 2A peptide is encoded by the following sequence: 5'-
  • polynucleotide sequence of the 2 A peptide may be modified or codon optimized by the methods described herein and/or are known in the art.
  • this sequence may be used to separate the coding regions of two or more polypeptides of interest.
  • the sequence encoding the F2A peptide may be between a first coding region A and a second coding region B (A-F2Apep-B).
  • the presence of the F2A peptide results in the cleavage of the one long protein between the glycine and the proline at the end of the F2A peptide sequence (NPGP is cleaved to result in NPG and P) thus creating separate protein A (with 21 amino acids of the F2A peptide attached, ending with NPG) and separate protein B (with 1 amino acid, P, of the F2A peptide attached).
  • Protein A and protein B may be the same or different peptides or polypeptides of interest (e.g., an ABCB4, ABCB11, or ATP8B1 polypeptide such as full length human ABCB4, ABCB11, or ATP8B1 or a truncated version thereof.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention is sequence optimized.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, optionally, a nucleotide sequence (e.g., an ORF) encoding another polypeptide of interest, a 5'-UTR, a 3'-UTR, the 5' UTR or 3' UTR optionally comprising at least one microRNA binding site, optionally a nucleotide sequence encoding a linker, a poly-A tail, or any combination thereof), in which the ORF(s) are sequence optimized.
  • a sequence-optimized nucleotide sequence e.g., a codon-optimized mRNA sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, is a sequence comprising at least one synonymous nucleobase substitution with respect to a reference sequence (e.g., a wild type nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide).
  • a sequence-optimized nucleotide sequence can be partially or completely different in sequence from the reference sequence.
  • a reference sequence encoding polyserine uniformly encoded by UCU codons can be sequence-optimized by having 100% of its nucleobases substituted (for each codon, U in position 1 replaced by A, C in position 2 replaced by G, and U in position 3 replaced by C) to yield a sequence encoding polyserine which would be uniformly encoded by AGC codons.
  • the percentage of sequence identity obtained from a global pairwise alignment between the reference polyserine nucleic acid sequence and the sequence-optimized polyserine nucleic acid sequence would be 0%.
  • the protein products from both sequences would be 100% identical.
  • sequence optimization also sometimes referred to codon optimization
  • results can include, e.g., matching codon frequencies in certain tissue targets and/or host organisms to ensure proper folding; biasing G/C content to increase mRNA stability or reduce secondary structures; minimizing tandem repeat codons or base runs that can impair gene construction or expression; customizing transcriptional and translational control regions; inserting or removing protein trafficking sequences; removing/adding post translation modification sites in an encoded protein (e.g., glycosylation sites); adding, removing or shuffling protein domains; inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; adjusting translational rates to allow the various domains of the protein to fold properly; and/or reducing or eliminating problem secondary structures within the polynucleotide.
  • Sequence optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • a sequence-optimized nucleotide sequence e.g., an ORF
  • the ABCB4, ABCB11, or ATP8B1 polypeptide, functional fragment, or a variant thereof encoded by the sequence-optimized nucleotide sequence has improved properties (e.g., compared to an ABCB4, ABCB11, or ATP8B1 polypeptide, functional fragment, or a variant thereof encoded by a reference nucleotide sequence that is not sequence optimized), e.g., improved properties related to expression efficacy after administration in vivo.
  • Such properties include, but are not limited to, improving nucleic acid stability (e.g., mRNA stability), increasing translation efficacy in the target tissue, reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, increasing and/or decreasing protein aggregation.
  • nucleic acid stability e.g., mRNA stability
  • increasing translation efficacy in the target tissue reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, increasing and/or decreasing protein aggregation.
  • sequence-optimized nucleotide sequence (e.g., an ORF) is codon optimized for expression in human subjects, having structural and/or chemical features that avoid one or more of the problems in the art, for example, features which are useful for optimizing formulation and delivery of nucleic acid-based therapeutics while retaining structural and functional integrity; overcoming a threshold of expression; improving expression rates; half-life and/or protein concentrations; optimizing protein localization; and avoiding deleterious bio-responses such as the immune response and/or degradation pathways.
  • an ORF codon optimized for expression in human subjects, having structural and/or chemical features that avoid one or more of the problems in the art, for example, features which are useful for optimizing formulation and delivery of nucleic acid-based therapeutics while retaining structural and functional integrity; overcoming a threshold of expression; improving expression rates; half-life and/or protein concentrations; optimizing protein localization; and avoiding deleterious bio-responses such as the immune response and/or degradation pathways.
  • the polynucleotides of the invention comprise a nucleotide sequence (e.g., a nucleotide sequence (e.g., an ORF) encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, a nucleotide sequence (e.g., an ORF) encoding another polypeptide of interest, a 5'-UTR, a 3'-UTR, a microRNA binding site, a nucleic acid sequence encoding a linker, or any combination thereof) that is sequence-optimized according to a method comprising: substituting at least one codon in a reference nucleotide sequence (e.g., an ORF encoding an ABCB4, ABCB11, or ATP8B1 polypeptide) with an alternative codon to increase or decrease uridine content to generate a uridine-modified sequence; substituting at least one codon in a reference nucleotide sequence (e.g., an ORF encoding an ABC
  • sequence-optimized nucleotide sequence e.g., an ORF encoding an ABCB4, ABCB11, or ATP8B1 polypeptide
  • the sequence optimization method is multiparametric and comprises one, two, three, four, or more methods disclosed herein and/or other optimization methods known in the art.
  • features which can be considered beneficial in some embodiments of the invention, can be encoded by or within regions of the polynucleotide and such regions can be upstream (5') to, downstream (3') to, or within the region that encodes the ABCB4, ABCB11, or ATP8B1 polypeptide. These regions can be incorporated into the polynucleotide before and/or after sequence-optimization of the protein encoding region or open reading frame (ORF). Examples of such features include, but are not limited to, untranslated regions (UTRs), microRNA sequences, Kozak sequences, oligo(dT) sequences, poly-A tail, and detectable tags and can include multiple cloning sites that can have Xbal recognition.
  • UTRs untranslated regions
  • microRNA sequences Kozak sequences
  • oligo(dT) sequences poly-A tail
  • detectable tags can include multiple cloning sites that can have Xbal recognition.
  • the polynucleotide of the invention comprises a 5' UTR, a 3' UTR and/or a microRNA binding site. In some embodiments, the polynucleotide comprises two or more 5' UTRs and/or 3' UTRs, which can be the same or different sequences. In some embodiments, the polynucleotide comprises two or more microRNA binding sites, which can be the same or different sequences. Any portion of the 5' UTR, 3' UTR, and/or microRNA binding site, including none, can be sequence-optimized and can independently contain one or more different structural or chemical modifications, before and/or after sequence optimization.
  • the polynucleotide is reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • the optimized polynucleotide can be reconstituted and transformed into chemically competent E. coli, yeast, neurospora, maize, drosophila, etc. where high copy plasmid-like or chromosome structures occur by methods described herein. Sequence-Optimized Nucleotide Sequences Encoding ABCB4, ABCB11, or ATP8B1 Polypeptides
  • the polynucleotide of the invention comprises a sequence- optimized nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide disclosed herein.
  • the polynucleotide of the invention comprises an open reading frame (ORF) encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, wherein the ORF has been sequence optimized.
  • Exemplary sequence-optimized nucleotide sequences encoding human ABCB4 are set forth as SEQ ID NOs: 68-92, 118, 120, 122, and 124.
  • Exemplary sequence-optimized nucleotide sequences encoding human ABCB11 are set forth as SEQ ID NOs: 126, 128, 130, 132, 134, 136, 138, 140, and 142.
  • Exemplary sequence-optimized nucleotide sequences encoding human ATP8B1 are set forth as SEQ ID NOs: 93-117.
  • the sequence optimized ABCB4, ABCB11, or ATP8B1 sequences, fragments, and variants thereof are used to practice the methods disclosed herein.
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, comprises from 5' to 3' end: a 5' cap provided herein, for example, Capl; a 5' UTR, such as the sequences provided herein, for example, SEQ ID NO: 12; an open reading frame encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, e.g., a sequence optimized nucleic acid sequence encoding ABCB4, ABCB11, or ATP8B1 set forth as SEQ ID NOs: 68-117, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, and 142; at least one stop codon (if not present at 5' terminus of 3 'UTR); a 3' UTR, such as the sequences provided here
  • all uracils in the polynucleotide are Nl-methylpseudouracil. In certain embodiments, all uracils in the polynucleotide are 5-methoxyuracil.
  • sequence-optimized nucleotide sequences disclosed herein are distinct from the corresponding wild type nucleotide acid sequences and from other known sequence- optimized nucleotide sequences, e.g., these sequence-optimized nucleic acids have unique compositional characteristics.
  • the percentage of uracil or thymine nucleobases in a sequence- optimized nucleotide sequence e.g., encoding an ABCB4, ABCB11, or ATP8B1
  • polypeptide, a functional fragment, or a variant thereof is modified (e.g., reduced) with respect to the percentage of uracil or thymine nucleobases in the reference wild-type nucleotide sequence.
  • a sequence is referred to as a uracil-modified or thymine-modified sequence.
  • the percentage of uracil or thymine content in a nucleotide sequence can be determined by dividing the number of uracils or thymines in a sequence by the total number of nucleotides and multiplying by 100.
  • the sequence-optimized nucleotide sequence has a lower uracil or thymine content than the uracil or thymine content in the reference wild-type sequence.
  • the uracil or thymine content in a sequence-optimized nucleotide sequence of the invention is greater than the uracil or thymine content in the reference wild-type sequence and still maintain beneficial effects, e.g., increased expression and/or reduced Toll-Like Receptor (TLR) response when compared to the reference wild-type sequence.
  • TLR Toll-Like Receptor
  • an ORF of any one or more of the sequences provided herein may be codon optimized.
  • Codon optimization in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
  • Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.
  • the open reading frame (ORF) sequence is optimized using optimization algorithms.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • a sequence optimized nucleic acid disclosed herein encoding an ABCB4, ABCB11, or ATP8B1 polypeptide can be tested to determine whether at least one nucleic acid sequence property (e.g., stability when exposed to nucleases) or expression property has been improved with respect to the non-sequence optimized nucleic acid.
  • expression property refers to a property of a nucleic acid sequence either in vivo (e.g., translation efficacy of a synthetic mRNA after administration to a subject in need thereof) or in vitro (e.g., translation efficacy of a synthetic mRNA tested in an in vitro model system).
  • Expression properties include but are not limited to the amount of protein produced by an mRNA encoding an ABCB4, ABCB11, or ATP8B1 polypeptide after administration, and the amount of soluble or otherwise functional protein produced.
  • sequence optimized nucleic acids disclosed herein can be evaluated according to the viability of the cells expressing a protein encoded by a sequence optimized nucleic acid sequence (e.g., a RNA, e.g., an mRNA) encoding an ABCB4, ABCB11, or ATP8B1 polypeptide disclosed herein.
  • a sequence optimized nucleic acid sequence e.g., a RNA, e.g., an mRNA
  • a plurality of sequence optimized nucleic acids disclosed herein e.g., a RNA, e.g., an mRNA
  • a property of interest for example an expression property in an in vitro model system, or in vivo in a target tissue or cell.
  • the desired property of the polynucleotide is an intrinsic property of the nucleic acid sequence.
  • the nucleotide sequence e.g., a RNA, e.g., an mRNA
  • the nucleotide sequence can be sequence optimized for in vivo or in vitro stability.
  • the nucleotide sequence can be sequence optimized for expression in a particular target tissue or cell.
  • the nucleic acid sequence is sequence optimized to increase its plasma half-life by preventing its degradation by endo and exonucleases.
  • the nucleic acid sequence is sequence optimized to increase its resistance to hydrolysis in solution, for example, to lengthen the time that the sequence optimized nucleic acid or a pharmaceutical composition comprising the sequence optimized nucleic acid can be stored under aqueous conditions with minimal degradation.
  • sequence optimized nucleic acid can be optimized to increase its resistance to hydrolysis in dry storage conditions, for example, to lengthen the time that the sequence optimized nucleic acid can be stored after lyophilization with minimal degradation.
  • the desired property of the polynucleotide is the level of expression of an ABCB4, ABCB11, or ATP8B1 polypeptide encoded by a sequence optimized sequence disclosed herein.
  • Protein expression levels can be measured using one or more expression systems.
  • expression can be measured in cell culture systems, e.g., CHO cells or HEK293 cells.
  • expression can be measured using in vitro expression systems prepared from extracts of living cells, e.g., rabbit reticulocyte lysates, or in vitro expression systems prepared by assembly of purified individual components.
  • the protein expression is measured in an in vivo system, e.g., mouse, rabbit, monkey, etc.
  • protein expression in solution form can be desirable.
  • a reference sequence can be sequence optimized to yield a sequence optimized nucleic acid sequence having optimized levels of expressed proteins in soluble form.
  • Levels of protein expression and other properties such as solubility, levels of aggregation, and the presence of truncation products (i.e., fragments due to proteolysis, hydrolysis, or defective translation) can be measured according to methods known in the art, for example, using electrophoresis (e.g., native or SDS-PAGE) or chromatographic methods (e.g., HPLC, size exclusion chromatography, etc.).
  • heterologous therapeutic proteins encoded by a nucleic acid sequence can have deleterious effects in the target tissue or cell, reducing protein yield, or reducing the quality of the expressed product (e.g., due to the presence of protein fragments or precipitation of the expressed protein in inclusion bodies), or causing toxicity.
  • sequence optimization of a nucleic acid sequence disclosed herein e.g., a nucleic acid sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid.
  • Heterologous protein expression can also be deleterious to cells transfected with a nucleic acid sequence for autologous or heterologous transplantation. Accordingly, in some embodiments of the present disclosure the sequence optimization of a nucleic acid sequence disclosed herein can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid sequence. Changes in cell or tissue viability, toxicity, and other physiological reaction can be measured according to methods known in the art. Reduction of Immune and/or Inflammatory Response
  • the administration of a sequence optimized nucleic acid encoding ABCB4, ABCB11, or ATP8B1 polypeptide or a functional fragment thereof can trigger an immune response, which could be caused by (i) the therapeutic agent (e.g., an mRNA encoding an ABCB4, ABCB11, or ATP8B1 polypeptide), or (ii) the expression product of such therapeutic agent (e.g., the ABCB4, ABCB11, or ATP8B1 polypeptide encoded by the mRNA), or (iv) a combination thereof.
  • the therapeutic agent e.g., an mRNA encoding an ABCB4, ABCB11, or ATP8B1 polypeptide
  • the expression product of such therapeutic agent e.g., the ABCB4, ABCB11, or ATP8B1 polypeptide encoded by the mRNA
  • sequence optimization of nucleic acid sequence can be used to decrease an immune or inflammatory response triggered by the administration of a nucleic acid encoding an ABCB4, ABCB11, or ATP8B1 polypeptide or by the expression product of ABCB4, ABCB11, or ATP8B1 encoded by such nucleic acid.
  • an inflammatory response can be measured by detecting increased levels of one or more inflammatory cytokines using methods known in the art, e.g., ELISA.
  • inflammatory cytokine refers to cytokines that are elevated in an inflammatory response.
  • inflammatory cytokines include interleukin-6 (IL-6), CXCL1 (chemokine (C-X-C motif) ligand 1; also known as GROa, interferon-g (IFNy), tumor necrosis factor a (TNFa), interferon g-induced protein 10 (PM0), or granulocyte-colony stimulating factor (G-CSF).
  • IFNy interleukin-6
  • CXCL1 chemokine (C-X-C motif) ligand 1
  • GROa interferon-g
  • TNFa tumor necrosis factor a
  • PM0 interferon g-induced protein 10
  • G-CSF granulocyte-colony stimulating factor
  • inflammatory cytokines includes also other cytokines associated with inflammatory responses known in the art, e.g., interleukin-l (IL-l), interleukin-8 (IL-8), interleukin- 12 (IL-12), interleukin- 13 (11-13), interferon a (IFN-a), etc.
  • IL-l interleukin-l
  • IL-8 interleukin-8
  • IL-12 interleukin- 12
  • IFN-a interferon a
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a chemically modified nucleobase, for example, a chemically modified uracil, e.g., pseudouracil, Nl-methylpseudouracil, 5-methoxyuracil, or the like.
  • the mRNA is a uracil-modified sequence comprising an ORF encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, wherein the mRNA comprises a chemically modified nucleobase, for example, a chemically modified uracil, e.g., pseudouracil,
  • Nl-methylpseudouracil or 5-methoxyuracil.
  • modified uracil in the polynucleotide is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, at least 95%, at least 99%, or about 100% modified uracil.
  • uracil in the polynucleotide is at least 95% modified uracil.
  • uracil in the polynucleotide is 100% modified uracil.
  • modified uracil content of the ORF is between about 100% and about 150%, between about 100% and about 110%, between about 105% and about 115%, between about 110% and about 120%, between about 115% and about 125%, between about 120% and about 130%, between about 125% and about 135%, between about 130% and about 140%, between about 135% and about 145%, between about 140% and about 150% of the theoretical minimum uracil content in the corresponding wild-type ORF (%UTM).
  • the uracil content of the ORF is between about 121% and about 136% or between 123% and 134% of the %UTM. In some embodiments, the uracil content of the ORF encoding an ABCB4, ABCB11, or ATP8B1 polypeptide is about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, or about 150% of the %UTM.
  • uracil can refer to modified uracil and/or naturally occurring uracil.
  • the uracil content in the ORF of the mRNA encoding an ABCB4, ABCB11, or ATP8B1 polypeptide of the invention is less than about 30%, about 25%, about 20%, about 15%, or about 10% of the total nucleobase content in the ORF. In some embodiments, the uracil content in the ORF is between about 10% and about 20% of the total nucleobase content in the ORF. In other embodiments, the uracil content in the ORF is between about 10% and about 25% of the total nucleobase content in the ORF.
  • the uracil content in the ORF of the mRNA encoding an ABCB4, ABCB11, or ATP8B1 polypeptide is less than about 20% of the total nucleobase content in the open reading frame.
  • uracil can refer to modified uracil and/or naturally occurring uracil.
  • the ORF of the mRNA encoding an ABCB4, ABCB11, or ATP8B1 polypeptide having modified uracil and adjusted uracil content has increased Cytosine (C), Guanine (G), or Guanine/Cytosine (G/C) content (absolute or relative).
  • the overall increase in C, G, or G/C content (absolute or relative) of the ORF is at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% relative to the G/C content (absolute or relative) of the wild-type ORF.
  • the G, the C, or the G/C content in the ORF is less than about 100%, less than about 90%, less than about 85%, or less than about 80% of the theoretical maximum G, C, or G/C content of the corresponding wild type nucleotide sequence encoding the ABCB4, ABCB11, or ATP8B1 polypeptide (%GTMX; %CTMX, or %G/CTMX).
  • the increases in G and/or C content (absolute or relative) described herein can be conducted by replacing synonymous codons with low G, C, or G/C content with synonymous codons having higher G, C, or G/C content.
  • the increase in G and/or C content (absolute or relative) is conducted by replacing a codon ending with U with a synonymous codon ending with G or C.
  • the ORF of the mRNA encoding an ABCB4, ABCB11, or ATP8B1 polypeptide of the invention comprises modified uracil and has an adjusted uracil content containing less uracil pairs (UU) and/or uracil triplets (UUU) and/or uracil quadruplets (UUUU) than the corresponding wild-type nucleotide sequence encoding the ABCB4, ABCB11, or ATP8B1 polypeptide.
  • the ORF of the mRNA encoding an ABCB4, ABCB11, or ATP8B1 polypeptide of the invention contains no uracil pairs and/or uracil triplets and/or uracil quadruplets. In some embodiments, uracil pairs and/or uracil triplets and/or uracil quadruplets are reduced below a certain threshold, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 occurrences in the ORF of the mRNA encoding the ABCB4, ABCB11, or ATP8B1 polypeptide.
  • a certain threshold e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 occurrences in the ORF of the mRNA encoding the ABCB4, ABCB11, or ATP8B1 polypeptide.
  • the ORF of the mRNA encoding the ABCB4, ABCB11, or ATP8B1 polypeptide of the invention contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-phenylalanine uracil pairs and/or triplets.
  • the ORF of the mRNA encoding the ABCB4, ABCB11, or ATP8B1 polypeptide contains no non-phenylalanine uracil pairs and/or triplets.
  • the ORF of the mRNA encoding an ABCB4, ABCB11, or ATP8B1 polypeptide of the invention comprises modified uracil and has an adjusted uracil content containing less uracil-rich clusters than the corresponding wild-type nucleotide sequence encoding the ABCB4, ABCB11, or ATP8B1 polypeptide.
  • the ORF of the mRNA encoding the ABCB4, ABCB11, or ATP8B1 polypeptide of the invention contains uracil-rich clusters that are shorter in length than corresponding uracil-rich clusters in the corresponding wild-type nucleotide sequence encoding the ABCB4, ABCB11, or ATP8B1 polypeptide.
  • alternative lower frequency codons are employed. At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% of the codons in the ABCB4, ABCB11, or ATP8B1 polypeptide encoding ORF of the modified uracil-comprising mRNA are substituted with alternative codons, each alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.
  • the ORF also has adjusted uracil content, as described above.
  • at least one codon in the ORF of the mRNA encoding the ABCB4, ABCB11, or ATP8B1 polypeptide is substituted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.
  • the adjusted uracil content, ABCB4, ABCB11, or ATP8B1 polypeptide-encoding ORF of the modified uracil-comprising mRNA exhibits expression levels of ABCB4, ABCB11, or ATP8B1 when administered to a mammalian cell that are higher than expression levels of ABCB4, ABCB11, or ATP8B1 from the corresponding wild- type mRNA.
  • the mammalian cell is a mouse cell, a rat cell, or a rabbit cell. In other embodiments, the mammalian cell is a monkey cell or a human cell.
  • the human cell is a HeLa cell, a BJ fibroblast cell, or a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • ABCB4, ABCB11, or ATP8B1 is expressed at a level higher than expression levels of ABCB4, ABCB11, or ATP8B1 from the corresponding wild-type mRNA when the mRNA is administered to a mammalian cell in vivo.
  • the mRNA is administered to mice, rabbits, rats, monkeys, or humans. In one embodiment, mice are null mice.
  • the mRNA is administered to mice in an amount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, or 0.2 mg/kg or about 0.5 mg/kg.
  • the mRNA is administered intravenously or intramuscularly.
  • the ABCB4, ABCB11, or ATP8B1 polypeptide is expressed when the mRNA is administered to a mammalian cell in vitro.
  • the expression is increased by at least about 2-fold, at least about 5-fold, at least about lO-fold, at least about 50-fold, at least about 500-fold, at least about 1500-fold, or at least about 3000-fold.
  • the expression is increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, 60%, about 70%, about 80%, about 90%, or about 100%.
  • adjusted uracil content, ABCB4, ABCB11, or ATP8B1 polypeptide-encoding ORF of the modified uracil-comprising mRNA exhibits increased stability.
  • the mRNA exhibits increased stability in a cell relative to the stability of a corresponding wild-type mRNA under the same conditions.
  • the mRNA exhibits increased stability including resistance to nucleases, thermal stability, and/or increased stabilization of secondary structure.
  • increased stability exhibited by the mRNA is measured by determining the half-life of the mRNA (e.g., in a plasma, serum, cell, or tissue sample) and/or determining the area under the curve (AUC) of the protein expression by the mRNA over time (e.g., in vitro or in vivo).
  • An mRNA is identified as having increased stability if the half-life and/or the AUC is greater than the half-life and/or the AUC of a corresponding wild-type mRNA under the same conditions.
  • the mRNA of the present invention induces a detectably lower immune response (e.g., innate or acquired) relative to the immune response induced by a corresponding wild-type mRNA under the same conditions.
  • the mRNA of the present disclosure induces a detectably lower immune response (e.g., innate or acquired) relative to the immune response induced by an mRNA that encodes for an ABCB4, ABCB11, or ATP8B1 polypeptide but does not comprise modified uracil under the same conditions, or relative to the immune response induced by an mRNA that encodes for an ABCB4, ABCB11, or ATP8B1 polypeptide and that comprises modified uracil but that does not have adjusted uracil content under the same conditions.
  • the innate immune response can be manifested by increased expression of pro-inflammatory cytokines, activation of intracellular PRRs (RIG-I, MDA5, etc.), cell death, and/or termination or reduction in protein translation.
  • a reduction in the innate immune response can be measured by expression or activity level of Type 1 interferons (e.g., IFN-a, IFN-b, IFN-K, IFN-d, IFN-e, IFN-t, IFN-co, and IFN-z) or the expression of interferon-regulated genes such as the toll-like receptors (e.g., TLR7 and TLR8), and/or by decreased cell death following one or more administrations of the mRNA of the invention into a cell.
  • Type 1 interferons e.g., IFN-a, IFN-b, IFN-K, IFN-d, IFN-e, IFN-t, IFN-co, and IFN-z
  • interferon-regulated genes such as the toll
  • the expression of Type-l interferons by a mammalian cell in response to the mRNA of the present disclosure is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% relative to a corresponding wild-type mRNA, to an mRNA that encodes an ABCB4, ABCB11, or ATP8B1 polypeptide but does not comprise modified uracil, or to an mRNA that encodes an ABCB4, ABCB11, or ATP8B1 polypeptide and that comprises modified uracil but that does not have adjusted uracil content.
  • the interferon is IFN-b.
  • cell death frequency caused by administration of mRNA of the present disclosure to a mammalian cell is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death frequency observed with a corresponding wild-type mRNA, an mRNA that encodes for an ABCB4, ABCB11, or ATP8B1 polypeptide but does not comprise modified uracil, or an mRNA that encodes for an ABCB4, ABCB11, or ATP8B1 polypeptide and that comprises modified uracil but that does not have adjusted uracil content.
  • the mammalian cell is a BJ fibroblast cell. In other embodiments, the mammalian cell is a splenocyte.
  • the mammalian cell is that of a mouse or a rat. In other embodiments, the mammalian cell is that of a human. In one embodiment, the mRNA of the present disclosure does not substantially induce an innate immune response of a mammalian cell into which the mRNA is introduced.
  • modified polynucleotides comprising a polynucleotide described herein (e.g., a polynucleotide, e.g. mRNA, comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide).
  • the modified polynucleotides can be chemically modified and/or structurally modified.
  • modified polynucleotides can be referred to as "modified polynucleotides.”
  • nucleosides and nucleotides of a polynucleotide e.g., RNA polynucleotides, such as mRNA polynucleotides
  • a “nucleoside” refers to a compound containing a sugar molecule (e.g, a pentose or ribose) or a derivative thereof in combination with an organic base (e.g, a purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase”).
  • A“nucleotide” refers to a nucleoside including a phosphate group.
  • Modified nucleotides can be synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.
  • Polynucleotides can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.
  • modified polynucleotides disclosed herein can comprise various distinct modifications.
  • the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications.
  • a modified polynucleotide, introduced to a cell can exhibit one or more desirable properties, e.g., improved protein expression, reduced immunogenicity, or reduced degradation in the cell, as compared to an unmodified polynucleotide.
  • a polynucleotide of the present invention e.g., a polynucleotide of the present invention
  • polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or
  • ATP8B1 polypeptide is structurally modified.
  • a "structural" modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG” can be chemically modified to "AT- 5meC-G". The same polynucleotide can be structurally modified from "ATCG" to
  • ATCCCG the dinucleotide "CC” has been inserted, resulting in a structural modification to the polynucleotide.
  • compositions of the present disclosure comprise, in some embodiments, at least one nucleic acid (e.g., RNA) having an open reading frame encoding ABCB4,
  • nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art.
  • nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides.
  • modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides.
  • modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.
  • a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art.
  • Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.
  • a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art.
  • Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177;
  • RNA e.g., mRNA
  • nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U).
  • nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT).
  • nucleic acids of the disclosure can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.
  • Nucleic acids of the disclosure e.g, DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids
  • Nucleic acids of the disclosure comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides.
  • a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.
  • a modified RNA nucleic acid e.g, a modified mRNA nucleic acid
  • introduced to a cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • a modified RNA nucleic acid (e.g, a modified mRNA nucleic acid), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g, a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • Nucleic acids e.g, RNA nucleic acids, such as mRNA nucleic acids
  • Nucleic acids in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties.
  • the modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars.
  • the modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.
  • the present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g ., RNA nucleic acids, such as mRNA nucleic acids).
  • A“nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g, a purine or pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”).
  • A“nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.
  • Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.
  • Modified nucleotide base pairing encompasses not only the standard adenosine- thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification.
  • non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil.
  • nucleic acids of the present disclosure Any combination of base/ sugar or linker may be incorporated into nucleic acids of the present disclosure.
  • modified nucleobases in nucleic acids comprise Nl -methyl -pseudouridine (m 1 y), 1 -ethyl- pseudouridine (e l ⁇
  • modified nucleobases in nucleic acids comprise 5-methoxymethyl uridine, 5-methylthio uridine, l-methoxym ethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy cytidine.
  • the polyribonucleotide includes a combination of at least two (e.g, 2, 3,
  • a RNA nucleic acid of the disclosure comprises Nl -methyl- pseudouridine (m 1 y) substitutions at one or more or all uridine positions of the nucleic acid.
  • a RNA nucleic acid of the disclosure comprises Nl -methyl- pseudouridine (m 1 y) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.
  • a RNA nucleic acid of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid.
  • a RNA nucleic acid of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.
  • a RNA nucleic acid of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.
  • nucleic acids e.g ., RNA nucleic acids, such as mRNA nucleic acids
  • RNA nucleic acids are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a nucleic acid can be uniformly modified with Nl -methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with Nl -methyl -pseudouridine.
  • a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.
  • nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule.
  • one or more or all or a given type of nucleotide e.g, purine or pyrimidine, or any one or more or all of A, G, U, C
  • nucleotides X in a nucleic acid of the present disclosure are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
  • the nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g, from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%
  • the nucleic acids may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.
  • the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine.
  • At least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g ., a 5-substituted uracil).
  • the modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • cytosine in the nucleic acid is replaced with a modified cytosine (e.g, a 5-substituted cytosine).
  • the modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g, 2, 3, 4 or more unique structures).
  • Translation of a polynucleotide comprising an open reading frame encoding a polypeptide can be controlled and regulated by a variety of mechanisms that are provided by various cis-acting nucleic acid structures.
  • cis-acting RNA elements that form hairpins or other higher-order (e.g., pseudoknot) intramolecular mRNA secondary structures can provide a translational regulatory activity to a polynucleotide, wherein the RNA element influences or modulates the initiation of polynucleotide translation, particularly when the RNA element is positioned in the 5' UTR close to the 5 '-cap structure (Pelletier and Sonenberg (1985) Cell 40(3):5l5-526; Kozak (1986) Proc Natl Acad Sci 83:2850-2854).
  • Untranslated regions are nucleic acid sections of a polynucleotide before a start codon (5' UTR) and after a stop codon (3' UTR) that are not translated.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA e.g., a messenger RNA (mRNA)
  • RNA messenger RNA
  • ORF open reading frame
  • ATP8B1 polypeptide further comprises UTR (e.g., a 5' UTR or functional fragment thereof, a 3' UTR or functional fragment thereof, or a combination thereof).
  • Cis-acting RNA elements can also affect translation elongation, being involved in numerous frameshifting events (Namy et al., (2004) Mol Cell 13(2): 157-168).
  • Internal ribosome entry sequences represent another type of cis-acting RNA element that are typically located in 5' UTRs, but have also been reported to be found within the coding region of naturally-occurring mRNAs (Holcik et al. (2000) Trends Genet 16(10):469-473).
  • IRES In cellular mRNAs, IRES often coexist with the 5 '-cap structure and provide mRNAs with the functional capacity to be translated under conditions in which cap-dependent translation is compromised (Gebauer et al, (2012) Cold Spring Harb Perspect Biol 4(7):a0l2245).
  • Another type of naturally-occurring cis-acting RNA element comprises upstream open reading frames (uORFs).
  • uORFs Naturally-occurring uORFs occur singularly or multiply within the 5' UTRs of numerous mRNAs and influence the translation of the downstream major ORF, usually negatively (with the notable exception of GCN4 mRNA in yeast and ATF4 mRNA in mammals, where uORFs serve to promote the translation of the downstream major ORF under conditions of increased eIF2 phosphorylation (Hinnebusch (2005) Annu Rev Microbiol 59:407-450)). Additional exemplary translational regulatory activities provided by components, structures, elements, motifs, and/or specific sequences comprising
  • polynucleotides include, but are not limited to, mRNA stabilization or destabilization (Baker & Parker (2004) Curr Opin Cell Biol l6(3):293-299), translational activation (Villalba et al., (2011) Curr Opin Genet Dev 2l(4):452-457), and translational repression (Blumer et al., (2002) Mech Dev 110(1 -2) : 97 -112).
  • mRNA stabilization or destabilization Boker & Parker (2004) Curr Opin Cell Biol l6(3):293-299
  • translational activation Villalba et al., (2011) Curr Opin Genet Dev 2l(4):452-457
  • translational repression Blumer et al., (2002) Mech Dev 110(1 -2) : 97 -112).
  • Studies have shown that naturally-occurring, cis-acting RNA elements can confer their respective functions when used to modify, by incorporation into, heterolog
  • the present disclosure provides synthetic polynucleotides comprising a modification (e.g., an RNA element), wherein the modification provides a desired translational regulatory activity.
  • the disclosure provides a polynucleotide comprising a 5' untranslated region (UTR), an initiation codon, a full open reading frame encoding a polypeptide, a 3' UTR, and at least one modification, wherein the at least one modification provides a desired translational regulatory activity, for example, a modification that promotes and/or enhances the translational fidelity of mRNA translation.
  • the desired translational regulatory activity is a cis-acting regulatory activity.
  • the desired translational regulatory activity is an increase in the residence time of the 43 S pre-initiation complex (PIC) or ribosome at, or proximal to, the initiation codon. In some embodiments, the desired translational regulatory activity is an increase in the initiation of polypeptide synthesis at or from the initiation codon. In some embodiments, the desired translational regulatory activity is an increase in the amount of polypeptide translated from the full open reading frame. In some embodiments, the desired translational regulatory activity is an increase in the fidelity of initiation codon decoding by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is inhibition or reduction of leaky scanning by the PIC or ribosome.
  • the desired translational regulatory activity is a decrease in the rate of decoding the initiation codon by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is inhibition or reduction in the initiation of polypeptide synthesis at any codon within the mRNA other than the initiation codon. In some embodiments, the desired translational regulatory activity is inhibition or reduction of the amount of polypeptide translated from any open reading frame within the mRNA other than the full open reading frame. In some embodiments, the desired translational regulatory activity is inhibition or reduction in the production of aberrant translation products. In some embodiments, the desired translational regulatory activity is a combination of one or more of the foregoing translational regulatory activities.
  • the present disclosure provides a polynucleotide, e.g., an mRNA, comprising an RNA element that comprises a sequence and/or an RNA secondary structure(s) that provides a desired translational regulatory activity as described herein.
  • the mRNA comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that promotes and/or enhances the translational fidelity of mRNA translation.
  • the mRNA comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that provides a desired translational regulatory activity, such as inhibiting and/or reducing leaky scanning.
  • the disclosure provides an mRNA that comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that inhibits and/or reduces leaky scanning thereby promoting the translational fidelity of the mRNA.
  • the RNA element comprises natural and/or modified nucleotides. In some embodiments, the RNA element comprises of a sequence of linked nucleotides, or derivatives or analogs thereof, that provides a desired translational regulatory activity as described herein. In some embodiments, the RNA element comprises a sequence of linked nucleotides, or derivatives or analogs thereof, that forms or folds into a stable RNA secondary structure, wherein the RNA secondary structure provides a desired translational regulatory activity as described herein.
  • RNA elements can be identified and/or characterized based on the primary sequence of the element (e.g., GC-rich element), by RNA secondary structure formed by the element (e.g.
  • RNA molecules e.g., located within the 5' UTR of an mRNA
  • biological function and/or activity of the element e.g.,“translational enhancer element”
  • the disclosure provides an mRNA having one or more structural modifications that inhibits leaky scanning and/or promotes the translational fidelity of mRNA translation, wherein at least one of the structural modifications is a GC-rich RNA element. In some aspects, the disclosure provides a modified mRNA comprising at least one
  • the GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5' UTR of the mRNA.
  • the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich RNA element is located 15-30, 15-20, 15- 25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence.
  • the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5 ' UTR of the mRNA.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%- 60% cytosine, 40-50% cytosine, 30-40% cytosine bases.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 3- 30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60% cytosine, 40-50% cytosine, or 30-40% cytosine.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14,
  • sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5' UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5' UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • sequence composition is >50% cytosine.
  • sequence composition is >55% cytosine, >60% cytosine, >65% cytosine, >70% cytosine, >75% cytosine, >80% cytosine, >85% cytosine, or >90% cytosine.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5' UTR of the mRNA, wherein the GC-rich RNA element comprises any one of the sequences set forth in Table 2.
  • the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich RNA element is located about 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence VI [CCCCGGCGCC (SEQ ID NO: 291)] as set forth in Table 2, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence VI as set forth in Table 2 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5'
  • the GC-rich element comprises the sequence VI as set forth in Table 2 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA. In other embodiments, the GC-rich element comprises the sequence VI as set forth in Table 2 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence V2 [CCCCGGC (SEQ ID NO:292)] as set forth in Table 2, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence V2 as set forth in Table 2 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5'
  • the GC-rich element comprises the sequence V2 as set forth in Table 2 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA. In other embodiments, the GC-rich element comprises the sequence V2 as set forth in Table 2 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence EK [GCCGCC (SEQ ID NO: 290)] as set forth in Table 2, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence EK as set forth in Table 2 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence EK as set forth in Table 2 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA. In other embodiments, the GC-rich element comprises the sequence EK as set forth in Table 2 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence VI [CCCCGGCGCC (SEQ ID NO: 291)] as set forth in Table 2, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5' UTR of the mRNA, wherein the 5' UTR comprises the following sequence shown in Table 2:
  • RNA sequences described herein will be Ts in a corresponding template DNA sequence, for example, in DNA templates or constructs from which mRNAs of the disclosure are transcribed, e.g., via IVT.
  • the GC-rich element comprises the sequence VI as set forth in Table 2 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5' UTR sequence shown in Table 2. In some embodiments, the GC-rich element comprises the sequence VI as set forth in Table 2 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA, wherein the 5' UTR comprises the following sequence shown in Table 2:
  • the GC-rich element comprises the sequence VI as set forth in Table 2 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA, wherein the 5' UTR comprises the following sequence shown in Table 2: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 12).
  • the 5' UTR comprises the following sequence set forth in Table 2:
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a stable RNA secondary structure comprising a sequence of nucleotides, or derivatives or analogs thereof, linked in an order which forms a hairpin or a stem-loop.
  • the stable RNA secondary structure is upstream of the Kozak consensus sequence.
  • the stable RNA secondary structure is located about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides upstream of the Kozak consensus sequence.
  • the stable RNA secondary structure is located about 20, about 15, about 10 or about 5 nucleotides upstream of the Kozak consensus sequence.
  • the stable RNA secondary structure is located about 5, about 4, about 3, about 2, about 1 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 15-30, about 15-20, about 15-25, about 10-15, or about 5-10 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located 12-15 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure has a deltaG of about -30 kcal/mol, about -20 to -30 kcal/mol, about -20 kcal/mol, about -10 to -20 kcal/mol, about -10 kcal/mol, about -5 to -10 kcal/mol.
  • the modification is operably linked to an open reading frame encoding a polypeptide and wherein the modification and the open reading frame are heterologous.
  • sequence of the GC-rich RNA element is comprised exclusively of guanine (G) and cytosine (C) nucleobases.
  • RNA elements that provide a desired translational regulatory activity as described herein can be identified and characterized using known techniques, such as ribosome profiling.
  • Ribosome profiling is a technique that allows the determination of the positions of PICs and/or ribosomes bound to mRNAs (see e.g., Ingolia et ah, (2009) Science
  • the technique is based on protecting a region or segment of mRNA, by the PIC and/or ribosome, from nuclease digestion.
  • RNA footprints can be analyzed by methods known in the art (e.g., RNA-seq). The footprint is roughly centered on the A-site of the ribosome. If the PIC or ribosome dwells at a particular position or location along an mRNA, footprints generated at these position would be relatively common. Studies have shown that more footprints are generated at positions where the PIC and/or ribosome exhibits decreased processivity and fewer footprints where the PIC and/or ribosome exhibits increased processivity (Gardin et ah, (2014) eLife 3:e03735). In some embodiments, residence time or the time of occupancy of the PIC or ribosome at a discrete position or location along a polynucleotide comprising any one or more of the RNA elements described herein is determined by ribosome profiling.
  • a UTR can be homologous or heterologous to the coding region in a polynucleotide.
  • the UTR is homologous to the ORF encoding the ABCB4, ABCB11, or ATP8B1 polypeptide.
  • the UTR is heterologous to the ORF encoding the ABCB4, ABCB11, or ATP8B1 polypeptide.
  • the polynucleotide comprises two or more 5' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.
  • polynucleotide comprises two or more 3' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.
  • the 5' UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof is sequence optimized.
  • the 5'UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., Nl-methylpseudouracil or 5-methoxyuracil.
  • UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization and/or translation efficiency.
  • a polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods.
  • a functional fragment of a 5' UTR or 3' UTR comprises one or more regulatory features of a full length 5' or 3' UTR, respectively.
  • Natural 5 UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG (SEQ ID NO:248), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G. 5' UTRs also have been known to form secondary structures that are involved in elongation factor binding.
  • liver-expressed mRNA such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, can enhance expression of polynucleotides in hepatic cell lines or liver.
  • 5'UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie-l, CD36), for myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF, CDl lb, MSR, Fr-l, i- NOS), for leukocytes (e.g., CD45, CD18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-A/B/C/D).
  • muscle e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin
  • endothelial cells e.g., Tie-l, CD36
  • myeloid cells e.g
  • UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, an encoded
  • polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development.
  • the UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
  • the 5' UTR and the 3' UTR can be heterologous.
  • the 5' UTR can be derived from a different species than the 3' UTR.
  • the 3' UTR can be derived from a different species than the 5' UTR.
  • Exemplary UTRs of the application include, but are not limited to, one or more 5 UTR and/or 3 UTR derived from the nucleic acid sequence of: a globin, such as an a- or b- globin (e.g., aXenopus , mouse, rabbit, or human globin); a strong Kozak translational initiation signal; a CYBA (e.g., human cytochro e b-245 a polypeptide); an albumin (e.g., human albumin7); a HSD17B4 (hydroxysteroid (17-b) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), a Sindbis virus,
  • C0I6AI C0I6AI
  • a ribophorin e.g., ribophorin I (RPNI)
  • RPNI ribophorin I
  • LRP1 low density lipoprotein receptor-related protein
  • a cardiotrophin-like cytokine factor e.g., Nntl
  • calreticulin Calr
  • Plodl procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1
  • nucleobindin e.g.,
  • the 5' UTR is selected from the group consisting of a b-globin 5' UTR; a 5 TR containing a strong Kozak translational initiation signal; a cytochrome b- 245 a polypeptide (CYBA) 5' UTR; a hydroxysteroid (17-b) dehydrogenase (HSD17B4) 5' UTR; a Tobacco etch virus (TEV) 5' UTR; a Vietnamese equine encephalitis virus (TEEV) 5' UTR; a 5' proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5' UTR; a heat shock protein 70 (Hsp70) 5' UTR; a eIF4G 5' UTR; a GLUT1 5' UTR; functional fragments thereof and any combination thereof.
  • CYBA cytochrome b- 245 a polypeptide
  • HSD17B4 hydroxysteroid
  • the 3' UTR is selected from the group consisting of a b-globin 3' UTR; a CYBA 3' UTR; an albumin 3 ' UTR; a growth hormone (GH) 3' UTR; a VEEV 3' UTR; a hepatitis B virus (HBV) 3' UTR; a-globin 3 UTR; a DEN 3' UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3' UTR; an elongation factor 1 al (EEF1A1) 3' UTR; a manganese superoxide dismutase (MnSOD) 3' UTR; a b subunit of mitochondrial H(+)-ATP synthase (b-mRNA) 3' UTR; a GLUT1 3' UTR; a MEF2A 3' UTR; a b-Fl-ATPase 3' UTR; functional fragments thereof and combinations thereof.
  • EEF1A1 manganese
  • Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the invention.
  • a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.
  • variants of 5' or 3' UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR.
  • one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3): 568-82, the contents of which are incorporated herein by reference in their entirety.
  • UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a 5' and/or 3' UTR can be inverted, shortened, lengthened, or combined with one or more other 5' UTRs or 3' UTRs.
  • the polynucleotide comprises multiple UTRs, e.g., a double, a triple or a quadruple 5' UTR or 3' UTR.
  • a double UTR comprises two copies of the same UTR either in series or substantially in series.
  • a double beta-globin 3 UTR can be used (see US2010/0129877, the contents of which are incorporated herein by reference in its entirety).
  • the polynucleotides of the invention comprise a 5' UTR and/or a 3' UTR selected from any of the UTRs disclosed herein.
  • the 5' UTR comprises any one of the exemplary 5’ UTR sequences presented below:
  • the 3' UTR comprises any one of the exemplary 3’ UTR sequences presented below:
  • the 5' UTR and/or 3' UTR sequence of the invention comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 5' UTR sequences comprising any of SEQ ID NO:3, 88-102, or 165-167 and/or 3' UTR sequences comprises any of SEQ ID NO:4, 104-112, or 150, and any combination thereof.
  • the polynucleotides of the invention can comprise combinations of features.
  • the ORF can be flanked by a 5 TR that comprises a strong Kozak translational initiation signal and/or a 3 UTR comprising an oligo(dT) sequence for templated addition of a poly-A tail.
  • a 5'UTR can comprise a first polynucleotide fragment and a second
  • polynucleotide fragment from the same and/or different UTRs (see, e.g., US2010/0293625, herein incorporated by reference in its entirety).
  • the polynucleotide of the invention comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010 394(1): 189-193, the contents of which are incorporated herein by reference in their entirety).
  • IRES internal ribosome entry site
  • the polynucleotide comprises an IRES instead of a 5' UTR sequence.
  • the polynucleotide comprises an ORF and a viral capsid sequence. In some embodiments, the polynucleotide comprises a synthetic 5' UTR in combination with a non-synthetic 3 ' UTR.
  • the UTR can also include at least one translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements
  • TEE refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide.
  • the TEE can be located between the transcription promoter and the start codon.
  • the 5' UTR comprises a TEE.
  • a TEE is a conserved element in a ETTR that can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation.
  • Polynucleotides of the invention can include regulatory elements, for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof.
  • miRNA microRNA
  • binding sites for example, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof.
  • polynucleotides including such regulatory elements are referred to as including “sensor sequences”.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • ORF open reading frame
  • miRNA binding site(s) provides for regulation of polynucleotides of the invention, and in turn, of the polypeptides encoded therefrom, based on tissue-specific and/or cell-type specific expression of naturally-occurring miRNAs.
  • compositions and formulations that comprise any of the polynucleotides described above.
  • the composition or formulation further comprises a delivery agent.
  • the composition or formulation can contain a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes a polypeptide. In some embodiments, the composition or formulation can contain a
  • polynucleotide e.g., a RNA, e.g., an mRNA
  • a polynucleotide e.g., an ORF
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds
  • a miRNA e.g., a natural-occurring miRNA
  • a miRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature miRNA.
  • a miRNA seed can comprise positions 2-8 or 2-7 of the mature miRNA.
  • microRNAs derive enzymatically from regions of RNA transcripts that fold back on themselves to form short hairpin structures often termed a pre-miRNA (precursor-miRNA).
  • a pre-miRNA typically has a two-nucleotide overhang at its 3' end, and has 3' hydroxyl and 5' phosphate groups.
  • This precursor-mRNA is processed in the nucleus and subsequently transported to the cytoplasm where it is further processed by DICER (a RNase III enzyme), to form a mature microRNA of approximately 22 nucleotides.
  • DICER a RNase III enzyme
  • the mature microRNA is then incorporated into a ribonuclear particle to form the RNA-induced silencing complex, RISC, which mediates gene silencing.
  • a miR referred to by number herein can refer to either of the two mature microRNAs originating from opposite arms of the same pre- miRNA (e.g., either the 3p or 5p microRNA). All miRs referred to herein are intended to include both the 3p and 5p arms/sequences, unless particularly specified by the 3p or 5p designation.
  • microRNA (miRNA or miR) binding site refers to a sequence within a polynucleotide, e.g., within a DNA or within an RNA transcript, including in the 5'UTR and/or 3'UTR, that has sufficient complementarity to all or a region of a miRNA to interact with, associate with or bind to the miRNA.
  • a polynucleotide of the invention comprising an ORF encoding a polypeptide of interest and further comprises one or more miRNA binding site(s).
  • a 5' UTR and/or 3' UTR of the polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • a miRNA binding site having sufficient complementarity to a miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated regulation of a polynucleotide, e.g., miRNA-mediated translational repression or degradation of the polynucleotide.
  • a miRNA binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the polynucleotide, e.g., miRNA-guided RNA- induced silencing complex (RISC)-mediated cleavage of mRNA.
  • miRNA-guided RNA- induced silencing complex RISC
  • the miRNA binding site can have complementarity to, for example, a 19-25 nucleotide long miRNA sequence, to a 19-23 nucleotide long miRNA sequence, or to a 22 nucleotide long miRNA sequence.
  • a miRNA binding site can be complementary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally-occurring miRNA sequence, or to a portion less than 1, 2, 3, or 4 nucleotides shorter than a naturally-occurring miRNA sequence.
  • Full or complete complementarity e.g., full complementarity or complete complementarity over all or a significant portion of the length of a naturally-occurring miRNA is preferred when the desired regulation is mRNA degradation.
  • a miRNA binding site includes a sequence that has
  • the miRNA binding site includes a sequence that has complete complementarity with a miRNA seed sequence.
  • a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with an miRNA sequence.
  • the miRNA binding site includes a sequence that has complete complementarity with a miRNA sequence.
  • a miRNA binding site has complete complementarity with a miRNA sequence but for 1, 2, or 3 nucleotide substitutions, terminal additions, and/or truncations.
  • the miRNA binding site is the same length as the
  • the miRNA binding site is one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve nucleotide(s) shorter than the corresponding miRNA at the 5' terminus, the 3' terminus, or both.
  • the microRNA binding site is two nucleotides shorter than the corresponding microRNA at the 5' terminus, the 3' terminus, or both. The miRNA binding sites that are shorter than the corresponding miRNAs are still capable of degrading the mRNA incorporating one or more of the miRNA binding sites or preventing the mRNA from translation.
  • the miRNA binding site binds the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA binding site to the corresponding miRNA in RISC degrades the mRNA containing the miRNA binding site or prevents the mRNA from being translated. In some embodiments, the miRNA binding site has sufficient complementarity to miRNA so that a RISC complex comprising the miRNA cleaves the polynucleotide comprising the miRNA binding site. In other embodiments, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA induces instability in the polynucleotide comprising the miRNA binding site. In another embodiment, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA represses transcription of the polynucleotide comprising the miRNA binding site.
  • the miRNA binding site has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) from the corresponding miRNA.
  • the miRNA binding site has at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one contiguous nucleotides complementary to at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one, respectively, contiguous nucleotides of the corresponding miRNA.
  • the polynucleotide By engineering one or more miRNA binding sites into a polynucleotide of the invention, the polynucleotide can be targeted for degradation or reduced translation, provided the miRNA in question is available. This can reduce off-target effects upon delivery of the polynucleotide. For example, if a polynucleotide of the invention is not intended to be delivered to a tissue or cell but ends up is said tissue or cell, then a miRNA abundant in the tissue or cell can inhibit the expression of the gene of interest if one or multiple binding sites of the miRNA are engineered into the 5' UTR and/or 3' UTR of the polynucleotide.
  • incorporation of one or more miRNA binding sites into an mRNA of the disclosure may reduce the hazard of off-target effects upon nucleic acid molecule delivery and/or enable tissue-specific regulation of expression of a polypeptide encoded by the mRNA.
  • incorporation of one or more miRNA binding sites into an mRNA of the disclosure can modulate immune responses upon nucleic acid delivery in vivo.
  • incorporation of one or more miRNA binding sites into an mRNA of the disclosure can modulate accelerated blood clearance (ABC) of lipid
  • miRNA binding sites can be removed from polynucleotide sequences in which they naturally occur in order to increase protein expression in specific tissues.
  • a binding site for a specific miRNA can be removed from a polynucleotide to improve protein expression in tissues or cells containing the miRNA.
  • Regulation of expression in multiple tissues can be accomplished through introduction or removal of one or more miRNA binding sites, e.g., one or more distinct miRNA binding sites.
  • the decision whether to remove or insert a miRNA binding site can be made based on miRNA expression patterns and/or their profilings in tissues and/or cells in development and/or disease. Identification of miRNAs, miRNA binding sites, and their expression patterns and role in biology have been reported (e.g., Bonauer et al., Curr Drug Targets 2010 11 :943-949; Anand and Cheresh Curr Opin Hematol 2011 18: 171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20. doi: 10.
  • tissues where miRNA are known to regulate mRNA, and thereby protein expression include, but are not limited to, liver (miR-l22), muscle (miR-l33, miR-206, miR- 208), endothelial cells (miR-l7-92, miR-l26), myeloid cells (miR-l42-3p, miR-l42-5p, miR- 16, miR-2l, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-ld, miR- 149), kidney (miR-l92, miR-l94, miR-204), and lung epithelial cells (let-7, miR-l33, miR- 126).
  • liver miR-l22
  • muscle miR-l33, miR-206, miR- 208
  • endothelial cells miR-l7-92, miR-l26
  • myeloid cells miR-l42-3p, miR
  • miRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc.
  • APCs antigen presenting cells
  • Immune cell specific miRNAs are involved in immunogenicity, autoimmunity, the immune-response to infection, inflammation, as well as unwanted immune response after gene therapy and tissue/organ transplantation. Immune cells specific miRNAs also regulate many aspects of development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells).
  • miR-l42 and miR-l46 are exclusively expressed in immune cells, particularly abundant in myeloid dendritic cells. It has been demonstrated that the immune response to a polynucleotide can be shut-off by adding miR- 142 binding sites to the 3'-UTR of the polynucleotide, enabling more stable gene transfer in tissues and cells. miR-l42 efficiently degrades exogenous polynucleotides in antigen presenting cells and suppresses cytotoxic elimination of transduced cells (e.g., Annoni A et ah, blood, 2009, 114, 5152-5161; Brown BD, et ah, Nat med. 2006, 12(5), 585-591; Brown BD, et ah, Blood, 2007, 110(13): 4144-4152, each of which is incorporated herein by reference in its entirety).
  • An antigen-mediated immune response can refer to an immune response triggered by foreign antigens, which, when entering an organism, are processed by the antigen presenting cells and displayed on the surface of the antigen presenting cells. T cells can recognize the presented antigen and induce a cytotoxic elimination of cells that express the antigen.
  • Introducing a miR-l42 binding site into the 5' UTR and/or 3'UTR of a polynucleotide of the invention can selectively repress gene expression in antigen presenting cells through miR-l42 mediated degradation, limiting antigen presentation in antigen presenting cells (e.g., dendritic cells) and thereby preventing antigen-mediated immune response after the delivery of the polynucleotide.
  • the polynucleotide is then stably expressed in target tissues or cells without triggering cytotoxic elimination.
  • binding sites for miRNAs that are known to be expressed in immune cells can be engineered into a polynucleotide of the invention to suppress the expression of the polynucleotide in antigen presenting cells through miRNA mediated RNA degradation, subduing the antigen-mediated immune response. Expression of the polynucleotide is maintained in non-immune cells where the immune cell specific miRNAs are not expressed.
  • any miR-l22 binding site can be removed and a miR-l42 (and/or mirR-l46) binding site can be engineered into the 5' UTR and/or 3' UTR of a polynucleotide of the invention.
  • a polynucleotide of the invention can include a further negative regulatory element in the 5' UTR and/or 3' UTR, either alone or in combination with miR-l42 and/or miR-l46 binding sites.
  • the further negative regulatory element is a Constitutive Decay Element (CDE).
  • Immune cell specific miRNAs include, but are not limited to, hsa-let-7a-2-3p, hsa-let- 7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa- let-7i-3p, hsa-let-7i-5p, miR-l0a-3p, miR-l0a-5p, miR-H84, hsa-let-7f-l— 3p, hsa-let-7f-2— 5p, hsa-let-7f-5p, miR-l25b-l-3p, miR-l25b-2-3p, miR-l25b-5p, miR-l279, miR-l30a-3p, miR-l30a-5p, miR-l32-3p, miR-l32-5
  • novel miRNAs can be identified in immune cell through micro-array hybridization and microtome analysis (e.g., Jima DD et al, Blood, 2010, 1 l6:el !8-el27; Vaz C et al., BMC Genomics, 2010, 11,288, the content of each of which is incorporated herein by reference in its entirety.)
  • miRNAs that are known to be expressed in the liver include, but are not limited to, miR-l07, miR-l22-3p, miR-l22-5p, miR-l228-3p, miR-l228-5p, miR-l249, miR-l29-5p, miR-l303, miR-l5la-3p, miR-l5la-5p, miR-l52, miR-l94-3p, miR-l94-5p, miR-l99a-3p, miR-l99a-5p, miR-l99b-3p, miR-l99b-5p, miR-296-5p, miR-557, miR-58l, miR-939-3p, and miR-939-5p. miRNA binding sites from any liver specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the
  • Liver specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a
  • miRNAs that are known to be expressed in the lung include, but are not limited to, let- 7a-2-3p, let-7a-3p, let-7a-5p, miR-l26-3p, miR-l26-5p, miR-l27-3p, miR-l27-5p, miR- l30a-3p, miR-l30a-5p, miR-l30b-3p, miR-l30b-5p, miR-l33a, miR-l33b, miR-l34, miR- l8a-3p, miR-l8a-5p, miR-l8b-3p, miR-l8b-5p, miR-24-l-5p, miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-38l-3p, and miR-38l- 5p.
  • miRNA binding sites from any lung specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the lung.
  • Lung specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the heart include, but are not limited to, miR-l, miR-l33a, miR-l33b, miR-l49-3p, miR-l49-5p, miR-l86-3p, miR-l86-5p, miR- 208a, miR-208b, miR-2lO, miR-296-3p, miR-320, miR-45la, miR-45lb, miR-499a-3p, miR- 499a-5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b- 5p.
  • miRNA binding sites from any heart specific microRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the heart.
  • Heart specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the nervous system include, but are not limited to, miR-l24-5p, miR-l25a-3p, miR-l25a-5p, miR-l25b-l-3p, miR-l25b-2-3p, miR- l25b-5p,miR-l27l-3p, miR-l27l-5p, miR-l28, miR-l32-5p, miR-l35a-3p, miR-l35a-5p, miR-l35b-3p, miR-l35b-5p, miR-l37, miR-l39-5p, miR-l39-3p, miR-l49-3p, miR-l49-5p, miR-l53, miR-l8lc-3p, miR-l8lc-5p, miR-l83-3p, miR-l83-5p, miR-l90a, miR-l90b, miR- 212-3r, miR-2l2
  • miRNAs enriched in the nervous system further include those specifically expressed in neurons, including, but not limited to, miR-l32-3p, miR-l32-3p, miR-l48b-3p, miR-l48b-5p, miR-l5la-3p, miR-l5la-5p, miR-2l2-3p, miR- 2l2-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328, miR-922 and those specifically expressed in glial cells, including, but not limited to, miR-l250, miR-2l9-l-3p, miR-2l9-2-3p, miR-2l9-5p, miR-23a-3p, miR-23a-5p, miR- 3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5
  • miRNA binding sites from any CNS specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the nervous system.
  • Nervous system specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the pancreas include, but are not limited to, miR-l05-3p, miR-l05-5p, miR-l84, miR-l95-3p, miR-l95-5p, miR-l96a-3p, miR-l96a- 5p, miR-2l4-3p, miR-2l4-5p, miR-2l6a-3p, miR-2l6a-5p, miR-30a-3p, miR-33a-3p, miR- 33a-5p, miR-375, miR-7-l-3p, miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944.
  • pancreas specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the pancreas.
  • Pancreas specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g. APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the kidney include, but are not limited to, miR-l22-3p, miR-l45-5p, miR-l7-5p, miR-l92-3p, miR-l92-5p, miR-l94-3p, miR-l94-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-2lO, miR-2l6a-3p, miR-2l6a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-l-3p, miR-30c- 2-3p, miR30c-5p, miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR-562.
  • kidney specific miRNA binding sites from any kidney specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the kidney.
  • Kidney specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the muscle include, but are not limited to, let-7g-3p, let-7g-5p, miR-l, miR-l286, miR-l33a, miR-l33b, miR-l40-3p, miR-l43-3p, miR-l43-5p, miR-l45-3p, miR-l45-5p, miR-l88-3p, miR-l88-5p, miR-206, miR-208a, miR- 208b, miR-25-3p, and miR-25-5p.
  • MiRNA binding sites from any muscle specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the muscle.
  • Muscle specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs are also differentially expressed in different types of cells, such as, but not limited to, endothelial cells, epithelial cells, and adipocytes.
  • miRNAs that are known to be expressed in endothelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-l00-3p, miR-l00-5p, miR-l0l-3p, miR-l0l-5p, miR- l26-3p, miR-l26-5p, miR-l236-3p, miR-l236-5p, miR-l30a-3p, miR-l30a-5p, miR-l7-5p, miR-l7-3p, miR-l8a-3p, miR-l8a-5p, miR-l9a-3p, miR-l9a-5p, miR-l9b-l-5p, miR-l9b-2- 5p, miR-l 9b-3p, miR-20a-3p, miR-20a-5p, miR-2l7, miR-2lO, miR-2l-3p, miR-2l-5p, miR- 22l-3p, mi
  • miRNA binding sites from any endothelial cell specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the endothelial cells.
  • miRNAs that are known to be expressed in epithelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-l246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-45la, miR-45lb, miR-494, miR- 802 and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific in respiratory ciliated epithelial cells, let-7 family, miR-l33a, miR-l33b, miR-l26 specific in lung epithelial cells, miR-382-3p, miR-382-5p specific in renal epithelial cells, and miR-762 specific in corneal epithelial cells.
  • miRNA binding sites from any epithelial cell specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the epithelial cells.
  • a large group of miRNAs are enriched in embryonic stem cells, controlling stem cell self-renewal as well as the development and/or differentiation of various cell lineages, such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells (e.g., Kuppusamy KT et al., Curr. Mol Med, 2013, 13(5), 757-764; Vidigal JA and Ventura A, Semin Cancer Biol.
  • miRNAs abundant in embryonic stem cells include, but are not limited to, let-7a-2- 3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p, miR-l03a-2-3p, miR-l03a-5p, miR-l06b-3p, miR-l06b-5p, miR-l246, miR-l275, miR-l38-l-3p, miR-l38-2-3p, miR-l38-5p, miR-l54- 3p, miR-l54-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-30la-3p, miR-30la-5p, miR-30la-5p, miR- 302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-302c-5p, miR-302d- 3p, miR-302d-5p, miR-302
  • miRNAs are selected based on expression and abundance in immune cells of the hematopoietic lineage, such as B cells, T cells, macrophages, dendritic cells, and cells that are known to express TLR7/ TLR8 and/or able to secrete cytokines such as endothelial cells and platelets.
  • the miRNA set thus includes miRs that may be responsible in part for the immunogenicity of these cells, and such that a corresponding miR-site incorporation in polynucleotides of the present invention (e.g., mRNAs) could lead to destabilization of the mRNA and/or suppression of translation from these mRNAs in the specific cell type.
  • Non-limiting representative examples include miR- 142, miR-l44, miR-l50, miR-l55 and miR-223, which are specific for many of the hematopoietic cells; miR-l42, miRl50, miR-l6 and miR-223, which are expressed in B cells; miR-223, miR-45l, miR-26a, miR-l6, which are expressed in progenitor hematopoietic cells; and miR-l26, which is expressed in plasmacytoid dendritic cells, platelets and endothelial cells.
  • tissue expression of miRs see e.g., Teruel -Montoya, R. et al.
  • Any one miR-site incorporation in the 3' UTR and/or 5' UTR may mediate such effects in multiple cell types of interest (e.g., miR-l42 is abundant in both B cells and dendritic cells).
  • polynucleotides of the invention contain two or more (e.g., two, three, four or more) miR bindings sites from: (i) the group consisting of miR-l42, miR-l44, miR-l50, miR-l55 and miR-223 (which are expressed in many hematopoietic cells); or (ii) the group consisting of miR-l42, miRl50, miR-l6 and miR-223 (which are expressed in B cells); or the group consisting of miR-223, miR-45l, miR-26a, miR-l6 (which are expressed in progenitor hematopoietic cells).
  • miR-l42, miR-l44, miR-l50, miR-l55 and miR-223 which are expressed in many hematopoietic cells
  • miR-l42, miRl50, miR-l6 and miR-223 which are expressed in B cells
  • miR-l42 and miR-l26 may also be beneficial to combine various miRs such that multiple cell types of interest are targeted at the same time (e.g., miR-l42 and miR-l26 to target many cells of the hematopoietic lineage and endothelial cells).
  • polynucleotides of the invention comprise two or more (e.g., two, three, four or more) miRNA bindings sites, wherein: (i) at least one of the miRs targets cells of the hematopoietic lineage (e.g., miR-l42, miR-l44, miR-l50, miR-l55 or miR-223) and at least one of the miRs targets plasmacytoid dendritic cells, platelets or endothelial cells (e.g., miR- 126); or (ii) at least one of the miRs targets B cells (e.g., miR-l42, miRl50, miR-l6 or miR- 223) and at least one of the miRs targets plasmacytoid dendritic cells, platelets or endothelial cells (e.g., miR-l26); or (iii) at least one of the miRs targets progenitor hematopo
  • the miRs targets cells of the hematopoietic lineage (e.g., miR-l42, miR-l44, miR-l50, miR-l55 or miR-223), at least one of the miRs targets B cells (e.g., miR-l42, miRl50, miR- 16 or miR-223) and at least one of the miRs targets plasmacytoid dendritic cells, platelets or endothelial cells (e.g., miR-l26); or any other possible combination of the foregoing four classes of miR binding sites (i.e., those targeting the hematopoietic lineage, those targeting B cells, those targeting progenitor hematopoietic cells and/or those targeting plas acytoid dendritic cells/platelets/endothelial cells).
  • miR-l26 cells of the hematopoietic lineage
  • B cells e.g., miR-l42, miRl50, mi
  • polynucleotides of the present invention can comprise one or more miRNA binding sequences that bind to one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines (e.g., in immune cells of peripheral lymphoid organs and/or splenocytes and/or endothelial cells).
  • miRNA binding sequences that bind to one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines (e.g., in immune cells of peripheral lymphoid organs and/or splenocytes and/or endothelial cells).
  • incorporation into an mRNA of one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro- inflammatory cytokines and/or chemokines reduces or inhibits immune cell activation (e.g., B cell activation, as measured by frequency of activated B cells) and/or cytokine production (e.g., production of IL-6, IFN-g and/or TNFa).
  • incorporation into an mRNA of one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro- inflammatory cytokines and/or chemokines can reduce or inhibit an anti-drug antibody (ADA) response against a protein of interest encoded by the mRNA.
  • ADA anti-drug antibody
  • polynucleotides of the invention can comprise one or more miR binding sequences that bind to one or more miRNAs expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines (e.g., in immune cells of peripheral lymphoid organs and/or splenocytes and/or endothelial cells).
  • incorporation into an mRNA of one or more miR binding sites reduces or inhibits accelerated blood clearance (ABC) of the lipid-comprising compound or composition for use in delivering the mRNA.
  • incorporation of one or more miR binding sites into an mRNA reduces serum levels of anti-PEG anti-IgM (e.g., reduces or inhibits the acute production of IgMs that recognize polyethylene glycol (PEG) by B cells) and/or reduces or inhibits proliferation and/or activation of plasmacytoid dendritic cells following administration of a lipid-comprising compound or composition comprising the mRNA.
  • serum levels of anti-PEG anti-IgM e.g., reduces or inhibits the acute production of IgMs that recognize polyethylene glycol (PEG) by B cells
  • PEG polyethylene glycol
  • miR sequences may correspond to any known microRNA expressed in immune cells, including but not limited to those taught in US Publication US2005/0261218 and US Publication US2005/0059005, the contents of which are incorporated herein by reference in their entirety.
  • Non-limiting examples of miRs expressed in immune cells include those expressed in spleen cells, myeloid cells, dendritic cells, plasmacytoid dendritic cells, B cells, T cells and/or macrophages.
  • miR-l42-3p, miR-l42-5p, miR-l6, miR-2l, miR-223, miR-24 and miR-27 are expressed in myeloid cells
  • miR-l55 is expressed in dendritic cells
  • miR-l46 is upregulated in macrophages upon TLR stimulation
  • miR-l26 is expressed in plasmacytoid dendritic cells.
  • the miR(s) is expressed abundantly or preferentially in immune cells.
  • miR-l42 miR-l42-3p and/or miR-l42-5p
  • miR-l26 miR-l26- 3p and/or miR-l26-5p
  • miR-l46 miR-l46-3p and/or miR-l46-5p
  • miR-l55 miR-l55- 3p and/or miRl55-5p
  • polynucleotides of the present invention comprise at least one microRNA binding site for a miR selected from the group consisting of miR-l42, miR- 146, miR- 155, miR- 126, miR- 16, miR-2l, miR-223, miR-24 and miR-27.
  • the mRNA comprises at least two miR binding sites for microRNAs expressed in immune cells.
  • the polynucleotide of the invention comprises 1-4, one, two, three or four miR binding sites for microRNAs expressed in immune cells.
  • the polynucleotide of the invention comprises three miR binding sites.
  • miR binding sites can be for microRNAs selected from the group consisting of miR- 142, miR- 146, miR- 155, miR- 126, miR- 16, miR-2l, miR-223, miR-24, miR-27, and combinations thereof.
  • the polynucleotide of the invention comprises two or more (e.g., two, three, four) copies of the same miR binding site expressed in immune cells, e.g., two or more copies of a miR binding site selected from the group of miRs consisting of miR- 142, miR- 146, miR- 155, miR- 126, miR- 16, miR-2l, miR-223, miR- 24, miR-27.
  • the polynucleotide of the invention comprises three copies of the same miRNA binding site.
  • use of three copies of the same miR binding site can exhibit beneficial properties as compared to use of a single miRNA binding site.
  • Non-limiting examples of sequences for 3' UTRs containing three miRNA bindings sites are shown in SEQ ID NO: 199 (three miR-l42-3p binding sites) and SEQ ID NO: 190 (three miR-l42-5p binding sites).
  • the polynucleotide of the invention comprises two or more (e.g., two, three, four) copies of at least two different miR binding sites expressed in immune cells.
  • Non-limiting examples of sequences of 3' UTRs containing two or more different miR binding sites are shown in SEQ ID NO: 173 (one miR-l42-3p binding site and one miR-l26- 3p binding site), SEQ ID NO: 179 (two miR-l42-5p binding sites and one miR-l42-3p binding sites), and SEQ ID NO: 182 (two miR-l55-5p binding sites and one miR-l42-3p binding sites).
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-l42-3p.
  • the polynucleotide of the invention comprises binding sites for miR-l42-3p and miR-l55 (miR-l55-3p or miR-l55-5p), miR- l42-3p and miR-l46 (miR-l46-3 or miR-l46-5p), or miR-l42-3p and miR-l26 (miR-l26-3p or miR-l26-5p).
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-l26-3p.
  • the polynucleotide of the invention comprises binding sites for miR-l26-3p and miR-l55 (miR-l55-3p or miR-l55-5p), miR- l26-3p and miR-l46 (miR-l46-3p or miR-l46-5p), or miR-l26-3p and miR-l42 (miR-l42- 3p or miR-l42-5p).
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-l42-5p.
  • the polynucleotide of the invention comprises binding sites for miR-l42-5p and miR-l55 (miR-l55-3p or miR-l55-5p), miR- l42-5p and miR-l46 (miR-l46-3 or miR-l46-5p), or miR-l42-5p and miR-l26 (miR-l26-3p or miR-l26-5p).
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-l55-5p.
  • the polynucleotide of the invention comprises binding sites for miR-l55-5p and miR-l42 (miR-l42-3p or miR-l42-5p), miR- 155-5p and miR-l46 (miR-l46-3 or miR-l46-5p), or miR-l55-5p and miR-l26 (miR-l26-3p or miR-l26-5p).
  • miRNA can also regulate complex biological processes such as angiogenesis (e.g., miR-l32) (Anand and Cheresh, Curr Opin Hematol 2011 18: 171-176).
  • angiogenesis e.g., miR-l32
  • Cheresh Curr Opin Hematol 2011 18: 171-176.
  • polynucleotides of the invention miRNA binding sites that are involved in such processes can be removed or introduced, in order to tailor the expression of the polynucleotides to biologically relevant cell types or relevant biological processes.
  • the polynucleotides of the invention are defined as auxotrophic polynucleotides.
  • a polynucleotide of the invention comprises a miRNA binding site, wherein the miRNA binding site comprises one or more nucleotide sequences selected from Table 3, including one or more copies of any one or more of the miRNA binding site sequences.
  • a polynucleotide of the invention further comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more of the same or different miRNA binding sites selected from Table 3, including any combination thereof.
  • the miRNA binding site binds to miR-l42 or is
  • the miR-l42 comprises SEQ ID NO: 144.
  • the miRNA binding site binds to miR-l42-3p or miR-l42-5p.
  • the miR-l42-3p binding site comprises SEQ ID NO: 146.
  • the miR-l42-5p binding site comprises SEQ ID NO: 148.
  • the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 146 or SEQ ID NO: 148.
  • the miRNA binding site binds to miR-l26 or is
  • the miR-l26 comprises SEQ ID NO: 149.
  • the miRNA binding site binds to miR-l26-3p or miR-l26-5p.
  • the miR-l26-3p binding site comprises SEQ ID NO: 151.
  • the miR-l26-5p binding site comprises SEQ ID NO: 153.
  • the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 151 or SEQ ID NO: 153.
  • the 3' UTR comprises two miRNA binding sites, wherein a first miRNA binding site binds to miR-l42 and a second miRNA binding site binds to miR-l26.
  • the 3' UTR binding to miR-l42 and miR-l26 comprises, consists, or consists essentially of the sequence of SEQ ID NO: 173 or 195.
  • a miRNA binding site is inserted in the polynucleotide of the invention in any position of the polynucleotide (e.g., the 5' UTR and/or 3' UTR).
  • the 5' UTR comprises a miRNA binding site.
  • the 3 ' UTR comprises a miRNA binding site.
  • the 5' UTR and the 3' UTR comprise a miRNA binding site.
  • the insertion site in the polynucleotide can be anywhere in the polynucleotide as long as the insertion of the miRNA binding site in the polynucleotide does not interfere with the translation of a functional polypeptide in the absence of the corresponding miRNA; and in the presence of the miRNA, the insertion of the miRNA binding site in the polynucleotide and the binding of the miRNA binding site to the corresponding miRNA are capable of degrading the polynucleotide or preventing the translation of the polynucleotide.
  • a miRNA binding site is inserted in at least about 30 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the invention comprising the ORF. In some embodiments, a miRNA binding site is inserted in at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at least about 100 nucle
  • a miRNA binding site is inserted in about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 90 nucleotides, about 30 nucleotides to about 80 nucleotides, about 40 nucleotides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 45 nucleotides to about 65 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the invention.
  • a miRNA binding site is inserted within the 3' UTR
  • a miRNA binding site is inserted immediately following the final stop codon.
  • a miRNA binding site is inserted further downstream of the stop codon, in which case there are 3 ' UTR bases between the stop codon and the miR binding site(s).
  • three non-limiting examples of possible insertion sites for a miR in a 3' UTR are shown in SEQ ID NOs: 183, 184, and 185, which show a 3' UTR sequence with a miR-l42-3p site inserted in one of three different possible insertion sites, respectively, within the 3 ' UTR.
  • one or more miRNA binding sites can be positioned within the 5' UTR at one or more possible insertion sites.
  • three non-limiting examples of possible insertion sites for a miR in a 5' UTR are shown in SEQ ID NOs: 187-189, which show a 5' UTR sequence with a miR-l42-3p site inserted into one of three different possible insertion sites, respectively, within the 5' UTR.
  • a codon optimized open reading frame encoding a polypeptide of interest comprises a stop codon and the at least one microRNA binding site is located within the 3' UTR 1-100 nucleotides after the stop codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a stop codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 3' UTR 30-50 nucleotides after the stop codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a stop codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 3' UTR at least 50 nucleotides after the stop codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a stop codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 3' UTR immediately after the stop codon, or within the 3' UTR 15-20 nucleotides after the stop codon or within the 3' UTR 70-80 nucleotides after the stop codon.
  • the 3' UTR comprises more than one miRNA binding site (e.g., 2-4 miRNA binding sites), wherein there can be a spacer region (e.g., of 10-100, 20-70 or 30-50 nucleotides in length) between each miRNA binding site.
  • the 3' UTR comprises a spacer region between the end of the miRNA binding site(s) and the poly-A tail nucleotides.
  • a spacer region of 10-100, 20-70 or 30-50 nucleotides in length can be situated between the end of the miRNA binding site(s) and the beginning of the poly-A tail.
  • a codon optimized open reading frame encoding a polypeptide of interest comprises a start codon and the at least one microRNA binding site is located within the 5' UTR 1-100 nucleotides before (upstream of) the start codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a start codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 5' UTR 10-50 nucleotides before (upstream of) the start codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a start codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 5' UTR at least 25 nucleotides before (upstream of) the start codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a start codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 5' UTR immediately before the start codon, or within the 5' UTR 15-20 nucleotides before the start codon or within the 5' UTR 70-80 nucleotides before the start codon.
  • the 5' UTR comprises more than one miRNA binding site (e.g., 2-4 miRNA binding sites), wherein there can be a spacer region (e.g., of 10-100, 20-70 or 30-50 nucleotides in length) between each miRNA binding site.
  • the 3' UTR comprises more than one stop codon, wherein at least one miRNA binding site is positioned downstream of the stop codons.
  • a 3' UTR can comprise 1, 2 or 3 stop codons.
  • triple stop codons that can be used include: UGAUAAUAG, UGAUAGUAA, UAAUGAUAG, UGAUAAUAA, UGAUAGUAG, UAAUGAUGA, UAAUAGUAG, UGAUGAUGA, UAAUAAUAA, and UAGUAGUAG.
  • 1, 2, 3 or 4 miRNA binding sites e.g., miR- l42-3p binding sites
  • these binding sites can be positioned directly next to each other in the construct (i.e., one after the other) or, alternatively, spacer nucleotides can be positioned between each binding site.
  • the 3' UTR comprises three stop codons with a single miR-l42- 3p binding site located downstream of the 3rd stop codon.
  • Non-limiting examples of sequences of 3' UTR having three stop codons and a single miR-l42-3p binding site located at different positions downstream of the final stop codon are shown in SEQ ID NOs: 172, 183-185. TABLE 4. 5' UTRs, 3'UTRs, miR sequences, and miR binding sites
  • miR l26-3p binding site bold underline
  • miR l42-5p binding site shaded and bold underline
  • the polynucleotide of the invention comprises a 5' UTR, a codon optimized open reading frame encoding a polypeptide of interest, a 3' UTR comprising the at least one miRNA binding site for a miR expressed in immune cells, and a 3' tailing region of linked nucleosides.
  • the 3' UTR comprises 1-4, at least two, one, two, three or four miRNA binding sites for miRs expressed in immune cells, preferably abundantly or preferentially expressed in immune cells.
  • the at least one miRNA expressed in immune cells is a miR-l42- 3p microRNA binding site.
  • the miR-l42-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 145. In one embodiment, the 3' UTR of the mRNA comprising the miR-l42-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 146.
  • the at least one miRNA expressed in immune cells is a miR-l26 microRNA binding site.
  • the miR-l26 binding site is a miR-l26-3p binding site.
  • the miR-l26-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 169.
  • the 3' UTR of the mRNA of the invention comprising the miR-l26-3p microRNA binding site comprises the sequence shown in SEQ ID NO: l5l.
  • Non-limiting exemplary sequences for miRs to which a microRNA binding site(s) of the disclosure can bind include the following: miR-l42-3p (SEQ ID NO: 145), miR-l42-5p (SEQ ID NO: 147), miR-l46-3p (SEQ ID NO: 155), miR-l46-5p (SEQ ID NO: 156), miR- 155-3p (SEQ ID NO: 157), miR-l55-5p (SEQ ID NO: 158), miR-l26-3p (SEQ ID NO: 150), miR-l26-5p (SEQ ID NO: 152), miR-l6-3p (SEQ ID NO: 159), miR-l6-5p (SEQ ID NO: 145), miR-l42-5p (SEQ ID NO: 147), miR-l46-3p (SEQ ID NO: 155), miR-l46-5p (SEQ ID NO: 156), miR- 155-3p (SEQ ID NO: 157),
  • miR-2l-3p SEQ ID NO: 161
  • miR-2l-5p SEQ ID NO: 162
  • miR-223-3p SEQ ID NO: 163
  • miR-223-5p SEQ ID NO: 164
  • miR-24-3p SEQ ID NO: 165
  • miR-24-5p SEQ ID NO: 166
  • miR-27-3p SEQ ID NO: 167
  • miR-27-5p SEQ ID NO: 168.
  • Other suitable miR sequences expressed in immune cells e.g., abundantly or preferentially expressed in immune cells
  • Sites that bind any of the aforementioned miRs can be designed based on Watson-Crick complementarity to the miR, typically 100% complementarity to the miR, and inserted into an mRNA construct of the disclosure as described herein.
  • a polynucleotide of the present invention (e.g., and mRNA, e.g., the 3' UTR thereof) can comprise at least one miRNA bindingsite to thereby reduce or inhibit accelerated blood clearance, for example by reducing or inhibiting production of IgMs, e.g., against PEG, by B cells and/or reducing or inhibiting proliferation and/or activation of pDCs, and can comprise at least one miRNA bindingsite for modulating tissue expression of an encoded protein of interest.
  • miRNA gene regulation can be influenced by the sequence surrounding the miRNA such as, but not limited to, the species of the surrounding sequence, the type of sequence (e.g., heterologous, homologous, exogenous, endogenous, or artificial), regulatory elements in the surrounding sequence and/or structural elements in the surrounding sequence.
  • the miRNA can be influenced by the 5'UTR and/or 3'UTR.
  • a non human 3 'UTR can increase the regulatory effect of the miRNA sequence on the expression of a polypeptide of interest compared to a human 3 ' UTR of the same sequence type.
  • other regulatory elements and/or structural elements of the 5' UTR can influence miRNA mediated gene regulation.
  • a regulatory element and/or structural element is a structured IRES (Internal Ribosome Entry Site) in the 5' UTR, which is necessary for the binding of translational elongation factors to initiate protein translation. EIF4A2 binding to this secondarily structured element in the 5'-UTR is necessary for miRNA mediated gene expression (Meijer HA et al., Science, 2013, 340, 82-85, herein incorporated by reference in its entirety).
  • the polynucleotides of the invention can further include this structured 5' UTR in order to enhance microRNA mediated gene regulation.
  • At least one miRNA binding site can be engineered into the 3' UTR of a
  • miRNA binding sites can be engineered into a 3' UTR of a polynucleotide of the invention.
  • 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1 miRNA binding sites can be engineered into the 3'UTR of a polynucleotide of the invention.
  • miRNA binding sites incorporated into a polynucleotide of the invention can be the same or can be different miRNA sites.
  • a combination of different miRNA binding sites incorporated into a polynucleotide of the invention can include combinations in which more than one copy of any of the different miRNA sites are incorporated.
  • miRNA binding sites incorporated into a polynucleotide of the invention can target the same or different tissues in the body.
  • tissue-, cell-type-, or disease-specific miRNA binding sites in the 3'-UTR of a polynucleotide of the invention the degree of expression in specific cell types (e.g., myeloid cells, endothelial cells, etc.) can be reduced.
  • a miRNA binding site can be engineered near the 5' terminus of the 3'UTR, about halfway between the 5' terminus and 3' terminus of the 3'UTR and/or near the 3' terminus of the 3' UTR in a polynucleotide of the invention.
  • a miRNA binding site can be engineered near the 5' terminus of the 3'UTR and about halfway between the 5' terminus and 3 ' terminus of the 3 UTR.
  • a miRNA binding site can be engineered near the 3' terminus of the 3'UTR and about halfway between the 5' terminus and 3' terminus of the 3' UTR.
  • a miRNA binding site can be engineered near the 5' terminus of the 3' UTR and near the 3 ' terminus of the 3 ' UTR.
  • a 3 'UTR can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA binding sites.
  • the miRNA binding sites can be complementary to a miRNA, miRNA seed sequence, and/or miRNA sequences flanking the seed sequence.
  • the expression of a polynucleotide of the invention can be controlled by incorporating at least one sensor sequence in the polynucleotide and
  • polynucleotide of the invention can be targeted to a tissue or cell by incorporating a miRNA binding site and formulating the polynucleotide in a lipid nanoparticle comprising an ionizable lipid, including any of the lipids described herein.
  • a polynucleotide of the invention can be engineered for more targeted expression in specific tissues, cell types, or biological conditions based on the expression patterns of miRNAs in the different tissues, cell types, or biological conditions.
  • tissue-specific miRNA binding sites Through introduction of tissue-specific miRNA binding sites, a polynucleotide of the invention can be designed for optimal protein expression in a tissue or cell, or in the context of a biological condition.
  • a polynucleotide of the invention can be designed to incorporate miRNA binding sites that either have 100% identity to known miRNA seed sequences or have less than 100% identity to miRNA seed sequences.
  • a polynucleotide of the invention can be designed to incorporate miRNA binding sites that have at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to known miRNA seed sequences.
  • the miRNA seed sequence can be partially mutated to decrease miRNA binding affinity and as such result in reduced downmodulation of the polynucleotide.
  • the degree of match or mismatch between the miRNA binding site and the miRNA seed can act as a rheostat to more finely tune the ability of the miRNA to modulate protein expression.
  • mutation in the non-seed region of a miRNA binding site can also impact the ability of a miRNA to modulate protein expression.
  • a miRNA sequence can be incorporated into the loop of a stem loop.
  • a miRNA seed sequence can be incorporated in the loop of a stem loop and a miRNA binding site can be incorporated into the 5' or 3' stem of the stem loop.
  • the miRNA sequence in the 5' UTR can be used to stabilize a polynucleotide of the invention described herein.
  • a miRNA sequence in the 5' UTR of a polynucleotide of the invention can be used to decrease the accessibility of the site of translation initiation such as, but not limited to a start codon. See, e.g., Matsuda et al., PLoS One.
  • a polynucleotide of the invention can comprise a miRNA sequence, instead of the LNA or EJC sequence described by Matsuda et al, near the site of translation initiation in order to decrease the accessibility to the site of translation initiation.
  • the site of translation initiation can be prior to, after or within the miRNA sequence.
  • the site of translation initiation can be located within a miRNA sequence such as a seed sequence or binding site.
  • a polynucleotide of the invention can include at least one miRNA in order to dampen the antigen presentation by antigen presenting cells.
  • the miRNA can be the complete miRNA sequence, the miRNA seed sequence, the miRNA sequence without the seed, or a combination thereof.
  • a miRNA can be the complete miRNA sequence, the miRNA seed sequence, the miRNA sequence without the seed, or a combination thereof.
  • a polynucleotide of the invention can be specific to the hematopoietic system.
  • a miRNA incorporated into a polynucleotide of the invention to dampen antigen presentation is miR-l42-3p.
  • a polynucleotide of the invention can include at least one miRNA in order to dampen expression of the encoded polypeptide in a tissue or cell of interest.
  • a polynucleotide of the invention can include at least one miR-l42-3p binding site, miR-l42-3p seed sequence, miR-l42-3p binding site without the seed, miR-l42-5p binding site, miR-l42-5p seed sequence, miR-l42-5p binding site without the seed, miR-l46 binding site, miR-l46 seed sequence and/or miR-l46 binding site without the seed sequence.
  • a polynucleotide of the invention can comprise at least one miRNA binding site in the 3 UTR in order to selectively degrade mRNA therapeutics in the immune cells to subdue unwanted immunogenic reactions caused by therapeutic delivery.
  • the miRNA binding site can make a polynucleotide of the invention more unstable in antigen presenting cells.
  • these miRNAs include miR-l42-5p, miR-l42-3p, miR-l46a-5p, and miR-l46-3p.
  • a polynucleotide of the invention comprises at least one miRNA sequence in a region of the polynucleotide that can interact with a RNA binding protein.
  • the polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • a RNA e.g., an mRNA
  • a sequence-optimized nucleotide sequence e.g., an ORF
  • an ABCB4, ABCB11, or ATP8B1 polypeptide e.g., the wild-type sequence, functional fragment, or variant thereof
  • a miRNA binding site e.g., a miRNA binding site that binds to miR-l42
  • miRNA binding site binds to miR-l26.
  • a polynucleotide of the present invention e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide of the invention
  • a polynucleotide of the present invention further comprises a 3' UTR.
  • 3'-UTR is the section of mRNA that immediately follows the translation termination codon and often contains regulatory regions that post-transcriptionally influence gene expression. Regulatory regions within the 3'-UTR can influence polyadenylation, translation efficiency, localization, and stability of the mRNA.
  • the 3'-UTR useful for the invention comprises a binding site for regulatory proteins or microRNAs.
  • the 3' UTR useful for the polynucleotides of the invention comprises a 3' UTR selected from the group consisting of SEQ ID NO: 13, 154, 170-173, 176-185, 190-222, or any combination thereof. In some embodiments, the 3' UTR selected from the group consisting of SEQ ID NO: 13, 206-222, or any combination thereof. In some embodiments, the 3' UTR comprises a nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the 3' UTR comprises a nucleic acid sequence of SEQ ID NO:206. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:207.
  • the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:208. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:209. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:2lO. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:2l 1. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:2l2. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:2l3.
  • the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:2l4. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:2l5. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:2l6. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:2l7. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:2l8. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:2l9.
  • the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:220. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:22l. In some embodiments, the 3' UTR comprises a nucleic acid sequences of SEQ ID NO:222.
  • the 3' UTR sequence useful for the invention comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 3' UTR sequences selected from the group consisting of SEQ ID NOs: 13, 154, 170-173, 176- 185, 190-222, or any combination thereof.
  • the disclosure also includes a polynucleotide that comprises both a 5' Cap and a polynucleotide of the present invention (e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide).
  • a polynucleotide that comprises both a 5' Cap and a polynucleotide of the present invention (e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide).
  • the 5' cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly-A binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the cap further assists the removal of 5' proximal introns during mRNA splicing.
  • Endogenous mRNA molecules can be 5 '-end capped generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule.
  • This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue.
  • the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5' end of the mRNA can optionally also be 2'-0- methylated.
  • 5 '-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.
  • the polynucleotides of the present invention e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or
  • ATP8B1 polypeptide incorporate a cap moiety.
  • polynucleotides of the present invention comprise a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified nucleotides can be used during the capping reaction.
  • a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) can be used with a- thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.
  • Additional modified guanosine nucleotides can be used such as a-m ethyl -phosphonate and seleno-phosphate nucleotides.
  • Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and/or 5 '-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxyl group of the sugar ring.
  • Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as a
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5'- caps in their chemical structure, while retaining cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the invention.
  • the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5 '-triphosphate-5 '-guanosine (m 7 G- 3'mppp-G; which can equivalently be designated 3' 0-Me-m7G(5')ppp(5')G).
  • the 3'-0 atom of the other, unmodified, guanine becomes linked to the 5 '-terminal nucleotide of the capped polynucleotide.
  • the N7- and 3 '-O-methlyated guanine provides the terminal moiety of the capped polynucleotide.
  • mCAP which is similar to ARCA but has a 2'-0-methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5 '-triphosphate-5 '-guanosine, m 7 Gm- ppp-G).
  • the cap is a dinucleotide cap analog.
  • the dinucleotide cap analog can be modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group such as the dinucleotide cap analogs described in U.S. Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety.
  • the cap is a cap analog is a N7-(4-chlorophenoxy ethyl) substituted dinucleotide form of a cap analog known in the art and/or described herein.
  • Non limiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and a N7-(4- chlorophenoxyethyl)-m 3 °G(5')ppp(5')G cap analog (See, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al.
  • a cap analog of the present invention is a 4- chloro/bromophenoxyethyl analog.
  • cap analogs allow for the concomitant capping of a polynucleotide or a region thereof, in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, can lead to reduced translational competency and reduced cellular stability.
  • Polynucleotides of the invention can also be capped post manufacture (whether IVT or chemical synthesis), using enzymes, in order to generate more authentic 5 '-cap structures.
  • the phrase "more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild- type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding
  • Non-limiting examples of more authentic 5 'cap structures of the present invention are those that, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5 'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild-type, natural or physiological 5'cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0- methyltransf erase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5'- terminal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5 '-terminal nucleotide of the mRNA contains a 2'-0- methyl.
  • Capl structure Such a structure is termed the Capl structure. This cap results in a higher
  • Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (cap 0),
  • capping chimeric polynucleotides post-manufacture can be more efficient as nearly 100% of the chimeric polynucleotides can be capped. This is in contrast to -80% when a cap analog is linked to a chimeric polynucleotide in the course of an in vitro transcription reaction.
  • 5' terminal caps can include endogenous caps or cap analogs.
  • a 5' terminal cap can comprise a guanine analog.
  • Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine.
  • the polynucleotides of the present disclosure e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or
  • ATP8B1 polypeptide further comprise a poly-A tail.
  • terminal groups on the poly-A tail can be incorporated for stabilization.
  • a poly- A tail comprises des-3' hydroxyl tails.
  • a long chain of adenine nucleotides can be added to a polynucleotide such as an mRNA molecule in order to increase stability.
  • poly-A polymerase adds a chain of adenine nucleotides to the RNA.
  • the process called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.
  • the poly-A tail is 100 nucleotides in length.
  • Poly-A tails can also be added after the construct is exported from the nucleus.
  • terminal groups on the poly-A tail can be incorporated for stabilization.
  • Polynucleotides of the present invention can include des-3 ' hydroxyl tails. They can also include structural moieties or 2'-Omethyl modifications as taught by Junjie Li, et al. (Current Biology, Vol. 15, 1501-1507, August 23, 2005, the contents of which are incorporated herein by reference in its entirety).
  • the polynucleotides of the present invention can be designed to encode transcripts with alternative poly-A tail structures including histone mRNA. According to Norbury, "Terminal uridylation has also been detected on human replication-dependent histone mRNAs. The turnover of these mRNAs is thought to be important for the prevention of potentially toxic histone accumulation following the completion or inhibition of chromosomal DNA replication.
  • mRNAs are distinguished by their lack of a 3' poly-A tail, the function of which is instead assumed by a stable stem-loop structure and its cognate stem-loop binding protein (SLBP); the latter carries out the same functions as those of PABP on polyadenylated mRNAs" (Norbury, "Cytoplas ic RNA: a case of the tail wagging the dog," Nature Reviews Molecular Cell Biology; AOP, published online 29 August 2013; doi: l0.l038/nrm3645) the contents of which are incorporated herein by reference in its entirety.
  • SLBP stem-loop binding protein
  • the length of a poly-A tail when present, is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length ( e.g ., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,
  • the polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to
  • the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design can be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides.
  • the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof.
  • the poly-A tail can also be designed as a fraction of the polynucleotides to which it belongs.
  • the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail.
  • engineered binding sites and conjugation of polynucleotides for Poly-A binding protein can enhance expression.
  • multiple distinct polynucleotides can be linked together via the PABP (Poly-A binding protein) through the 3 '-end using modified nucleotides at the 3 '-terminus of the poly-A tail.
  • Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at l2hr, 24hr, 48hr, 72hr and day 7 post transfection.
  • the polynucleotides of the present invention are designed to include a poly-A-G quartet region.
  • the G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA.
  • the G-quartet is incorporated at the end of the poly-A tail.
  • the resultant polynucleotide is assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the poly-A-G quartet results in protein production from an mRNA equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone.
  • the invention also includes a polynucleotide that comprises both a start codon region and the polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide).
  • a polynucleotide that comprises both a start codon region and the polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide).
  • the polynucleotides of the present invention can have regions that are analogous to or function like a start codon region.
  • the translation of a polynucleotide can initiate on a codon that is not the start codon AUG.
  • Translation of the polynucleotide can initiate on an alternative start codon such as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG,
  • the translation of a polynucleotide begins on the alternative start codon ACG.
  • polynucleotide translation begins on the alternative start codon CTG or CUG.
  • the translation of a polynucleotide begins on the alternative start codon GTG or GUG.
  • Nucleotides flanking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect the translation efficiency, the length and/or the structure of the polynucleotide. (See, e.g., Matsuda and Mauro PLoS ONE, 2010 5: 11; the contents of which are herein incorporated by reference in its entirety).
  • Masking any of the nucleotides flanking a codon that initiates translation can be used to alter the position of translation initiation, translation efficiency, length and/or structure of a polynucleotide.
  • a masking agent can be used near the start codon or alternative start codon in order to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon.
  • masking agents include antisense locked nucleic acids (LNA) polynucleotides and exon- junction complexes (EJCs) (See, e.g, Matsuda and Mauro describing masking agents LNA polynucleotides and EJCs (PLoS ONE, 2010 5: 11); the contents of which are herein incorporated by reference in its entirety).
  • a masking agent can be used to mask a start codon of a polynucleotide in order to increase the likelihood that translation will initiate on an alternative start codon.
  • a masking agent can be used to mask a first start codon or alternative start codon in order to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.
  • a start codon or alternative start codon can be located within a perfect complement for a miRNA binding site.
  • the perfect complement of a miRNA binding site can help control the translation, length and/or structure of the polynucleotide similar to a masking agent.
  • the start codon or alternative start codon can be located in the middle of a perfect complement for a miRNA binding site.
  • the start codon or alternative start codon can be located after the first nucleotide, second nucleotide, third nucleotide, fourth nucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide, eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventh nucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenth nucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenth nucleotide, eighteenth nucleotide, nineteenth nucleotide, twentieth nucleotide or twenty-first nucleotide.
  • the start codon of a polynucleotide can be removed from the polynucleotide sequence in order to have the translation of the polynucleotide begin on a codon that is not the start codon.
  • Translation of the polynucleotide can begin on the codon following the removed start codon or on a downstream start codon or an alternative start codon.
  • the start codon ATG or AEiG is removed as the first 3 nucleotides of the polynucleotide sequence in order to have translation initiate on a downstream start codon or alternative start codon.
  • the polynucleotide sequence where the start codon was removed can further comprise at least one masking agent for the downstream start codon and/or alternative start codons in order to control or attempt to control the initiation of translation, the length of the polynucleotide and/or the structure of the polynucleotide.
  • the invention also includes a polynucleotide that comprises both a stop codon region and the polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide).
  • the polynucleotides of the present invention can include at least two stop codons before the 3' untranslated region (UTR).
  • the stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA.
  • the polynucleotides of the present invention include the stop codon TGA in the case or DNA, or the stop codon UGA in the case of RNA, and one additional stop codon.
  • the addition stop codon can be TAA or UAA.
  • the polynucleotides of the present invention include three consecutive stop codons, four stop codons, or more.
  • Polynucleotide Comprising an niRXA Encoding an ABCB4, ABCB11, or ATP8B1
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, comprises from 5' to 3' end: a 5' cap provided above; a 5' UTR, such as the sequences provided above; an open reading frame encoding an ABCB4, ABCB11, or ATP8B1 polypeptide, e.g., a sequence optimized nucleic acid sequence encoding an ABCB4, ABCB11, or ATP8B1 disclosed herein; at least one stop codon; a 3' UTR, such as the sequences provided above; and a poly-A tail provided above.
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miRNA- 142.
  • the 5' UTR comprises the miRNA binding site.
  • the 3' UTR comprises the miRNA binding site.
  • a polynucleotide of the present disclosure comprises a nucleotide sequence encoding a polypeptide sequence at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% identical to the protein sequence of a wild type human ABCB4 (SEQ ID NO: 1), wild type human ABCB11 (SEQ ID NO:7), or wild type human ATP8B1 (SEQ ID NO:9).
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding a polypeptide, comprises (1) a 5' cap provided above, for example, CAP1, (2) a 5' UTR, (3) a nucleotide sequence ORF selected from the group consisting of SEQ ID NO:2, 5-8, 10-13, 15-18, or 20-23, (3) a stop codon, (4) a 3 'UTR, and (5) a poly-A tail provided above, for example, a poly-A tail of about 100 residues.
  • SEQ ID NO: 14 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 15, and 3 ' UTR of SEQ ID NO: 13.
  • SEQ ID NO: 17 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 18, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:20 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 21, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:23 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 24, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:26 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 27, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:29 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 30, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:32 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 33, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:35 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 36, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:38 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: l2, ABCB4 nucleotide ORF of SEQ ID NO: 39, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:4l consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 42, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:44 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: l2, ABCB4 nucleotide ORF of SEQ ID NO:45, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:47 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 48, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:50 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 51, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:53 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 54, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:56 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 57, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:59 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 60, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:62 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 63, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:65 consists from 5' to 3 ' end: 5' UTR of SEQ ID NO: 12, ABCB4 nucleotide ORF of SEQ ID NO: 66, and 3' UTR of SEQ ID NO: 13.
  • ABCB11 nucleotide constructs are described below:
  • SEQ ID NO: 126 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ABCB11 nucleotide ORF of SEQ ID NO: 249, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO: 128 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ABCB11 nucleotide ORF of SEQ ID NO:250, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO: 130 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ABCB11 nucleotide ORF of SEQ ID NO: 251, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO: 132 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ABCB11 nucleotide ORF of SEQ ID NO:252, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO: 134 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ABCB11 nucleotide ORF of SEQ ID NO:253, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO: 136 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ABCB11 nucleotide ORF of SEQ ID NO:254, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO: 138 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ABCB11 nucleotide ORF of SEQ ID NO:255, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO: 140 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ABCB11 nucleotide ORF of SEQ ID NO:256, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO: 142 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ABCB11 nucleotide ORF of SEQ ID NO:257, and 3' UTR of SEQ ID NO: 13.
  • ATP8B1 nucleotide constructs are described below:
  • SEQ ID NO:258 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 93, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:259 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 94, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:260 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 95, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:26l consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 96, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:262 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 97, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:263 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 98, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:264 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 99, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:265 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 100, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:266 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 101, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:267 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 102, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:268 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 103, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:269 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 104, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:270 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO : 105 , and 3 ' UTR of SEQ ID NO : 13.
  • SEQ ID NO:27l consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO : 106, and 3 ' UTR of SEQ ID NO : 13.
  • SEQ ID NO:272 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO : 107, and 3 ' UTR of SEQ ID NO : 13.
  • SEQ ID NO:273 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 108, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:274 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 109, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:275 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 110, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:276 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 111, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:277 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 112, and 3 ' UTR of SEQ ID NO: 13.
  • SEQ ID NO:278 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 113, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:279 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 114, and 3 ' UTR of SEQ ID NO: 13.
  • SEQ ID NO:280 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 115, and 3 ' UTR of SEQ ID NO: 13.
  • SEQ ID NO:28l consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 116, and 3' UTR of SEQ ID NO: 13.
  • SEQ ID NO:282 consists from 5' to 3' end: 5' UTR of SEQ ID NO: 12, ATP8B1 nucleotide ORF of SEQ ID NO: 117, and 3' UTR of SEQ ID NO: 13.
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding a ABCB4, ABCB11, or ATP8B1 polypeptide, comprises (1) a 5' cap provided above, for example, CAP1, (2) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20, 23, 26,
  • the present disclosure also provides methods for making a polynucleotide of the invention (e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide) or a complement thereof.
  • a polynucleotide of the invention e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • IVT in vitro transcription
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • encoding an ABCB4, ABCB11, or ATP8B1 polypeptide can be constructed by chemical synthesis using an oligonucleotide synthesizer.
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • encoding an ABCB4, ABCB11, or ATP8B1 polypeptide is made by using a host cell.
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • encoding an ABCB4, ABCB11, or ATP8B1 polypeptide is made by one or more combination of the IVT, chemical synthesis, host cell expression, or any other methods known in the art.
  • Naturally occurring nucleosides non-naturally occurring nucleosides, or
  • RNA e.g., an mRNA
  • ATP8B1 polypeptide e.g., an ABCB4, ABCB11, or ATP8B1 polypeptide.
  • the resultant polynucleotides, e.g., mRNAs can then be examined for their ability to produce protein and/or produce a therapeutic outcome.
  • polynucleotides of the present invention disclosed herein can be transcribed using an in vitro transcription (IVT) system.
  • IVT in vitro transcription
  • the system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.
  • NTPs can be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.
  • the polymerase can be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate polynucleotides disclosed herein. See U.S. Publ. No. US20130259923, which is herein incorporated by reference in its entirety.
  • RNA polymerases can be modified by inserting or deleting amino acids of the RNA polymerase sequence.
  • the RNA polymerase can be modified to exhibit an increased ability to incorporate a 2 '-modified nucleotide triphosphate compared to an unmodified RNA polymerase (see International Publication W02008078180 and U.S. Patent 8,101,385; herein incorporated by reference in their entireties).
  • Variants can be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and/or nucleic acid sequence and/or by using other methods known in the art.
  • T7 RNA polymerase variants can be evolved using the continuous directed evolution system set out by Esvelt et al.
  • T7 RNA polymerase can encode at least one mutation such as, but not limited to, lysine at position 93 substituted for threonine (K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, ATP8B122K, S228A, Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A, H523
  • T7 RNA polymerase variants can encode at least mutation as described in ET.S. Pub. Nos. 20100120024 and 20070117112; herein incorporated by reference in their entireties.
  • Variants of RNA polymerase can also include, but are not limited to, substitutional variants, conservative amino acid substitution, insertional variants, and/or deletional variants.
  • the polynucleotide can be designed to be recognized by the wild type or variant RNA polymerases. In doing so, the polynucleotide can be modified to contain sites or regions of sequence changes from the wild type or parent chimeric polynucleotide.
  • Polynucleotide or nucleic acid synthesis reactions can be carried out by enzymatic methods utilizing polymerases.
  • Polymerases catalyze the creation of phosphodiester bonds between nucleotides in a polynucleotide or nucleic acid chain.
  • DNA polymerase I polymerase I
  • a polymerase family including the Klenow fragments of E. coli , Bacillus DNA polymerase I, Thermus aquaticus (Taq) DNA polymerases, and the T7 RNA and DNA polymerases, is among the best studied of these families.
  • DNA polymerase a or B polymerase family, including all eukaryotic replicating DNA polymerases and polymerases from phages T4 and RB69. Although they employ similar catalytic mechanism, these families of polymerases differ in substrate specificity, substrate analog-incorporating efficiency, degree and rate for primer extension, mode of DNA synthesis, exonuclease activity, and sensitivity against inhibitors.
  • DNA polymerases are also selected based on the optimum reaction conditions they require, such as reaction temperature, pH, and template and primer concentrations.
  • RNA polymerases from bacteriophage T3, T7, and SP6 have been widely used to prepare RNAs for biochemical and biophysical studies. RNA polymerases, capping enzymes, and poly-A polymerases are disclosed in the co-pending International Publication No. WO2014/028429, the contents of which are incorporated herein by reference in their entirety.
  • the RNA polymerase which can be used in the synthesis of the polynucleotides of the present invention is a Syn5 RNA polymerase (see Zhu et al. Nucleic Acids Research 2013, doi: 10. l093/nar/gktl 193, which is herein incorporated by reference in its entirety).
  • the Syn5 RNA polymerase was recently characterized from marine cyanophage Syn5 by Zhu et al. where they also identified the promoter sequence (see Zhu et al. Nucleic Acids Research 2013, the contents of which is herein incorporated by reference in its entirety). Zhu et al.
  • Syn5 RNA polymerase catalyzed RNA synthesis over a wider range of temperatures and salinity as compared to T7 RNA polymerase. Additionally, the requirement for the initiating nucleotide at the promoter was found to be less stringent for Syn5 RNA polymerase as compared to the T7 RNA polymerase making Syn5 RNA polymerase promising for RNA synthesis.
  • a Syn5 RNA polymerase can be used in the synthesis of the polynucleotides described herein.
  • a Syn5 RNA polymerase can be used in the synthesis of the polynucleotide requiring a precise 3 '-terminus.
  • a Syn5 promoter can be used in the synthesis of the polynucleotides.
  • the Syn5 promoter can be 5 -ATTGGGCACCCGTAAGGG-3' (SEQ ID NO:283 as described by Zhu et al. (Nucleic Acids Research 2013).
  • RNA polymerase in the synthesis of
  • polynucleotides comprising at least one chemical modification described herein and/or known in the art (see e.g., the incorporation of pseudo-UTP and 5Me-CTP described in Zhu et al. Nucleic Acids Research 2013).
  • the polynucleotides described herein can be synthesized using a Syn5 RNA polymerase which has been purified using modified and improved purification procedure described by Zhu et al. (Nucleic Acids Research 2013).
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence-based amplification
  • TMA rolling-circle amplification
  • RCA rolling-circle amplification
  • Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest, such as a polynucleotide of the invention (e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide).
  • a polynucleotide of the invention e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide.
  • a single DNA or RNA oligomer containing a codon- optimized nucleotide sequence coding for the particular isolated polypeptide can be synthesized.
  • several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated.
  • the individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • a polynucleotide disclosed herein e.g., a RNA, e.g., an mRNA
  • a RNA e.g., an mRNA
  • a polynucleotide disclosed herein can be chemically synthesized using chemical synthesis methods and potential nucleobase substitutions known in the art. See, for example, International Publication Nos. WO2014093924,
  • Purification of the polynucleotides described herein can include, but is not limited to, polynucleotide clean-up, quality assurance and quality control.
  • Clean-up can be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc., Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • AGENCOURT® beads Beckman Coulter Genomics, Danvers, MA
  • poly-T beads poly-T beads
  • LNATM oligo-T capture probes EXIQON® Inc., Vedbaek, Denmark
  • HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • purified when used in relation to a polynucleotide such as a “purified polynucleotide” refers to one that is separated from at least one contaminant.
  • a "contaminant” is any substance that makes another unfit, impure or inferior.
  • a purified polynucleotide e.g., DNA and RNA
  • purification of a polynucleotide of the invention e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or
  • ATP8B1 polypeptide removes impurities that can reduce or remove an unwanted immune response, e.g, reducing cytokine activity.
  • the polynucleotide of the invention e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide
  • column chromatography e.g, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)
  • the polynucleotide of the invention e.g., a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide
  • column chromatography e.g, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC, hydrophobic interaction HPLC (HIC- HPLC), or (LCMS)
  • RP-HPLC reverse phase HPLC
  • HIC- HPLC hydrophobic interaction HPLC
  • LCMS hydrophobic interaction HPLC
  • a column chromatography e.g, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)
  • purified polynucleotide comprises a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide comprising one or more of the point mutations known in the art.
  • the use of RP-HPLC purified polynucleotide increases ABCB4, ABCB11, or ATP8B1 protein expression levels in cells when introduced into those cells, e.g., by 10-100%, i.e., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% with respect to the expression levels of ABCB4, ABCB11, or ATP8B1 protein in the cells before the RP-HPLC purified polynucleotide was introduced in the cells, or after a non-RP- HPLC purified polynucleotide was introduced in the cells.
  • the use of RP-HPLC purified polynucleotide increases functional ABCB4, ABCB11, or ATP8B1 protein expression levels in cells when introduced into those cells, e.g, by 10-100%, i.e., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% with respect to the functional expression levels of ABCB4, ABCB11, or ATP8B1 protein in the cells before the RP-HPLC purified polynucleotide was introduced in the cells, or after a non-RP-HPLC purified polynucleotide was introduced in the cells.
  • the use of RP-HPLC purified polynucleotide increases detectable ABCB4, ABCB11, or ATP8B1 activity in cells when introduced into those cells, e.g, by 10-100%, i.e., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% with respect to the activity levels of functional ABCB4, ABCB11, or ATP8B1 in the cells before the RP-HPLC purified polynucleotide was introduced in the cells, or after a non-RP-HPLC purified polynucleotide was introduced in the cells.
  • the purified polynucleotide is at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, at least about 96% pure, at least about 97% pure, at least about 98% pure, at least about 99% pure, or about 100% pure.
  • a quality assurance and/or quality control check can be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.
  • the polynucleotide can be sequenced by methods including, but not limited to reverse-transcriptase-PCR.
  • the polynucleotides of the present invention e.g., a
  • polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or
  • ATP8B1 polypeptide their expression products, as well as degradation products and metabolites can be quantified according to methods known in the art.
  • the polynucleotides of the present invention can be quantified in exosomes or when derived from one or more bodily fluid.
  • bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities,
  • exosomes can be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.
  • exosome quantification method a sample of not more than 2mL is obtained from the subject and the exosomes isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration,
  • the level or concentration of a polynucleotide can be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker.
  • the assay can be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes can be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods.
  • Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • the polynucleotide can be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).
  • UV/Vis ultraviolet visible spectroscopy
  • a non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA).
  • the quantified polynucleotide can be analyzed in order to determine if the polynucleotide can be of proper size, check that no degradation of the polynucleotide has occurred.
  • Degradation of the polynucleotide can be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
  • HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
  • compositions and formulations that comprise any of the polynucleotides described above.
  • the composition or formulation further comprises a delivery agent.
  • the composition or formulation can contain a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes an ABCB4, ABCB11, or ATP8B1 polypeptide.
  • the composition or formulation can contain a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes an ABCB4 polypeptide, a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes an ABCB11 polypeptide, and a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes an ATP8B1 polypeptide.
  • the composition or formulation can contain a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a polynucleotide (e.g., an ORF) having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes an ABCB4, ABCB11, or ATP8B1 polypeptide.
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • an ORF a polynucleotide having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes an ABCB4, ABCB11, or ATP8B1 polypeptide.
  • the composition or formulation can contain a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a polynucleotide (e.g., an ORF) having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes an ABCB4 polypeptide, a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a polynucleotide (e.g., an ORF) having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes an ABCB11 polypeptide, and a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a polynucleotide (e.g., an ORF) having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes an ATP8B1 polypeptide.
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds miR-l26, miR-l42, miR-l44, miR-l46, miR-l50, miR-l55, miR-l6, miR-2l, miR- 223, miR-24, miR-27 and miR-26a.
  • a miRNA binding site e.g., a miRNA binding site that binds miR-l26, miR-l42, miR-l44, miR-l46, miR-l50, miR-l55, miR-l6, miR-2l, miR- 223, miR-24, miR-27 and miR-26a.
  • compositions or formulation can optionally comprise one or more additional active substances, e.g., therapeutically and/or prophylactically active substances.
  • Pharmaceutical compositions or formulation of the present invention can be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of
  • compositions are administered to humans, human patients or subjects.
  • active ingredient generally refers to polynucleotides to be delivered as described herein.
  • Formulations and pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology.
  • such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition or formulation in accordance with the present disclosure can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure can vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • compositions and formulations described herein can contain at least one polynucleotide of the invention.
  • the composition or formulation can contain 1, 2, 3, 4 or 5 polynucleotides of the invention.
  • the compositions or formulations described herein can comprise more than one type of polynucleotide.
  • the composition or formulation can comprise a polynucleotide in linear and circular form. In another embodiment, the
  • composition or formulation can comprise a circular polynucleotide and an in vitro transcribed (IVT) polynucleotide.
  • IVT in vitro transcribed
  • the composition or formulation can comprise an IVT polynucleotide, a chimeric polynucleotide and a circular polynucleotide.
  • compositions and formulations are principally directed to pharmaceutical compositions and formulations that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals.
  • the present invention provides pharmaceutical formulations that comprise one or more polynucleotides described herein (e.g., one or more polynucleotides comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide).
  • the polynucleotides described herein can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g, from a depot formulation of the polynucleotide); (4) alter the biodistribution (e.g, target the polynucleotide to specific tissues or cell types); (5) increase the translation of encoded protein in vivo ; and/or (6) alter the release profile of encoded protein in vivo.
  • excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g, from a depot formulation of the polynucleotide); (4) alter the biodistribution (e.g, target the polynucleotide to specific tissues or cell types); (5) increase the translation of encoded protein in vivo ; and/or (6) alter the release profile of encoded protein in vivo.
  • the pharmaceutical formulation further comprises a delivery agent comprising, e.g., a compound having the Formula (I), e.g., any of Compounds 1-232, e.g., Compound II; a compound having the Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound VI; or a compound having the Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound I, or any combination thereof.
  • a delivery agent comprising, e.g., a compound having the Formula (I), e.g., any of Compounds 1-232, e.g., Compound II; a compound having the Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound VI; or a compound having the Formula (VIII), e.g., any of Compound
  • the delivery agent comprises Compound II, DSPC, Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio of about 50: 10:38.5: 1.5. In some embodiments, the delivery agent comprises Compound II, DSPC, Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio of about 47.5: 10.5:39.0:3.0. In some embodiments, the delivery agent comprises Compound VI, DSPC, Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio of about 50: 10:38.5: 1.5. In some embodiments, the delivery agent comprises Compound VI, DSPC, Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio of about
  • a pharmaceutically acceptable excipient includes, but are not limited to, any and all solvents, dispersion media, or other liquid vehicles, dispersion or suspension aids, diluents, granulating and/or dispersing agents, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, binders, lubricants or oil, coloring, sweetening or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmolality adjusting agents, pH adjusting agents, buffers, chelants, cyoprotectants, and/or bulking agents, as suited to the particular dosage form desired.
  • Exemplary diluents include, but are not limited to, calcium or sodium carbonate, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, etc., and/or combinations thereof.
  • Exemplary granulating and/or dispersing agents include, but are not limited to, starches, pregelatinized starches, or microcrystalline starch, alginic acid, guar gum, agar, poly(vinyl-pyrrolidone), (providone), cross-linked poly(vinyl-pyrrolidone) (crospovidone), cellulose, methylcellulose, carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, etc., and/or combinations thereof.
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], glyceryl monooleate, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ®30]), PLUORINC®F 68, POLOXAMER®l88, etc. and/or combinations thereof.
  • natural emulsifiers e.g.,
  • Exemplary binding agents include, but are not limited to, starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic gums (e.g., acacia, sodium alginate), ethyl cellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, etc., and combinations thereof.
  • sugars e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol
  • amino acids e.g., glycine
  • natural and synthetic gums e.g., acacia, sodium alginate
  • ethyl cellulose hydroxyethylcellulose, hydroxypropyl methylcellulose, etc., and combinations thereof.
  • Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations.
  • antioxidants can be added to the formulations.
  • Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium or potassium metabi sulfite, propionic acid, propyl gallate, sodium ascorbate, etc., and combinations thereof.
  • Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, trisodium edetate, etc., and combinations thereof.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • fumaric acid malic acid
  • phosphoric acid sodium edetate
  • tartaric acid trisodium edetate, etc.
  • antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, etc., and combinations thereof.
  • Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, butylated hydroxyanisol, ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), etc., and combinations thereof.
  • the pH of polynucleotide solutions is maintained between pH 5 and pH 8 to improve stability.
  • exemplary buffers to control pH can include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, etc., and/or combinations thereof.
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium or magnesium lauryl sulfate, etc., and combinations thereof.
  • the pharmaceutical composition or formulation described here can contain a cryoprotectant to stabilize a polynucleotide described herein during freezing.
  • Exemplary cryoprotectants include, but are not limited to mannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc., and combinations thereof.
  • the pharmaceutical composition or formulation described here can contain a bulking agent in lyophilized polynucleotide formulations to yield a "pharmaceutically elegant" cake, stabilize the lyophilized polynucleotides during long term (e.g., 36 month) storage.
  • exemplary bulking agents of the present invention can include, but are not limited to sucrose, trehalose, mannitol, glycine, lactose, raffmose, and combinations thereof.
  • the pharmaceutical composition or formulation further comprises a delivery agent.
  • the delivery agent of the present disclosure can include, without limitation, liposomes, lipid nanoparticles, lipidoids, polymers, lipoplexes, microvesicles, exosomes, peptides, proteins, cells transfected with polynucleotides, hyaluronidase, nanoparticle mimics, nanotubes, conjugates, and combinations thereof.
  • the present disclosure provides pharmaceutical compositions with advantageous properties.
  • the lipid compositions described herein may be advantageously used in lipid nanoparticle compositions for the delivery of therapeutic and/or prophylactic agents, e.g., mRNAs, to mammalian cells or organs.
  • the lipids described herein have little or no immunogenicity.
  • the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA).
  • a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent, e.g., mRNA, has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g, MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.
  • a reference lipid e.g, MC3, KC2, or DLinDMA
  • the present application provides pharmaceutical
  • compositions comprising: a polynucleotide comprising a nucleotide sequence encoding an ABCB4, ABCB11, or ATP8B1 polypeptide; and a delivery agent.
  • the present application provides pharmaceutical
  • compositions comprising: a polynucleotide comprising a nucleotide sequence encoding an ABCB4 polypeptide, a polynucleotide comprising a nucleotide sequence encoding an ABCB11 polypeptide, and a polynucleotide comprising a nucleotide sequence encoding an ATP8B1 polypeptide; and a delivery agent.
  • nucleic acids of the invention are formulated in a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • Lipid nanoparticles typically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest.
  • the lipid nanoparticles of the invention can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551;
  • PCT/US2015/027400 PCT/US2016/047406; PCT/US2016000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.
  • Nucleic acids of the present disclosure are typically formulated in lipid nanoparticle.
  • the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid.
  • PEG polyethylene glycol
  • the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid.
  • the lipid nanoparticle may comprise a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60% ionizable cationic lipid.
  • the lipid nanoparticle comprises a molar ratio of 20%, 30%, 40%, 50, or 60% ionizable cationic lipid.
  • the lipid nanoparticle comprises a molar ratio of 5-25% non- cationic lipid.
  • the lipid nanoparticle may comprise a molar ratio of 5-20%, 5- 15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, or 20-25% non-cationic lipid.
  • the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or 25% non-cationic lipid.
  • the lipid nanoparticle comprises a molar ratio of 25-55% sterol.
  • the lipid nanoparticle may comprise a molar ratio of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% sterol.
  • the lipid nanoparticle comprises a molar ratio of 25%, 30%, 35%, 40%, 45%, 50%, or 55% sterol.
  • the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG-modified lipid.
  • the lipid nanoparticle may comprise a molar ratio of 0.5- 10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%.
  • the lipid nanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEG-modified lipid.
  • the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.
  • the ionizable lipids of the present disclosure may be one or more of compounds of Formula (I):
  • Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
  • R 2 and R 3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of hydrogen, a C 3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R) 2 , and unsubstituted Ci- 6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -0(CH 2 ) n N(R) 2 , -C(0)0R, -0C(0)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -N(R) 2 , -C(0)N(R) 2 , -N(R)C(0)R, -N(R)S(0) 2 R, -N(R)C(0)N(R) 2 , -N(R)C(S)N(R) 2 , -N(R)R 8 , -N(R)S(0) 2 R 8 , -0(CH 2 )
  • n is independently selected from 1, 2, 3, 4, and 5;
  • each R 5 is independently selected from the group consisting of Ci -3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of Ci -3 alkyl, C 2-3 alkenyl, and H;
  • M and M’ are independently selected from -C(0)0-, -OC(O)-, -0C(0)-M”-C(0)0-, -C(0)N(R’)-, -N(R’)C(0)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(0)(0R’)0-, -S(0) 2 -, -S-S-, an aryl group, and a heteroaryl group, in which M” is a bond, Ci-i 3 alkyl or C 2 -i 3 alkenyl;
  • R 7 is selected from the group consisting of Ci -3 alkyl, C 2-3 alkenyl, and H;
  • R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, N0 2 , Ci- 6 alkyl, -OR, -S(0) 2 R, -S(0) 2 N(R) 2 , C 2-6 alkenyl, C3-6 carbocycle and heterocycle;
  • each R is independently selected from the group consisting of Ci- 3 alkyl, C 2-3 alkenyl, and H;
  • each R’ is independently selected from the group consisting of C1-18 alkyl, C 2 -i 8 alkenyl, -R*YR”, -YR”, and H;
  • each R is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl
  • each R* is independently selected from the group consisting of Ci-i 2 alkyl and C 2-i 2 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and wherein when R 4 is -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, or -CQ(R) 2 , then (i) Q is not -N(R) 2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.
  • a subset of compounds of Formula (I) includes those of Formula (IA):
  • R 4 is hydrogen, unsubstituted C1-3 alkyl, or -(CFh) n Q, in which Q is OH, -NHC(S)N(R) 2 , -NHC(0)N(R) 2 , -N(R)C(0)R,
  • -N(R)C(0)OR heteroaryl or heterocycloalkyl
  • M and M’ are independently selected from -C(0)0-, -OC(O)-, -0C(0)-M”-C(0)0-, -C(0)N(R , -P(0)(OR’)0-, -S-S-, an aryl group, and a heteroaryl group
  • R 2 and R3 are independently selected from the group consisting of H, Ci-i 4 alkyl, and C 2-i4 alkenyl.
  • m is 5, 7, or 9.
  • Q is OH, -NHC(S)N(R) 2 , or -NHC(0)N(R) 2 .
  • Q is -N(R)C(0)R, or -N(R)S(0) 2 R.
  • a subset of compounds of Formula (I) includes those of Formula (IB): (IB), or its N-oxide, or a salt or isomer thereof in which all variables are as defined herein.
  • m is selected from 5, 6, 7, 8, and 9;
  • R 4 is hydrogen, unsubstituted Ci- 3 alkyl, or -(CH 2 ) n Q, in which Q is OH, -NHC(S)N(R) 2 ,
  • -NHC( CHR 9 )N(R) 2 , -0C(0)N(R) 2 , -N(R)C(0)0R, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(0)0-, -OC(O)-, -0C(0)-M”-C(0)0-, -C(0)N(R , -P(0)(0R’)0-, -S-S-, an aryl group, and a heteroaryl group; and R 2 and R 3 are independently selected from the group consisting of H, Ci-i 4 alkyl, and C 2-i4 alkenyl.
  • m is 5, 7, or 9.
  • Q is OH, -NHC(S)N(R) 2 , or -NHC(0)N(R) 2 .
  • Q is -N(R)C(0)R, or -N(R)S(0) 2 R.
  • a subset of compounds of Formula (I) includes those of Formula (II):
  • M and M’ are independently selected from -C(0)0-, -OC(O)-, -0C(0)-M”-C(0)0-, -C(0)N(R , -P(0)(0R’)0-, -S-S-, an aryl group, and a heteroaryl group; and R 2 and Ri are independently selected from the group consisting of H, C l-l4 alkyl, and C 2.l4 alkenyl.
  • the compounds of Formula (I) are of Formula (Ha),
  • the compounds of Formula (I) are of Formula (lib),
  • the compounds of Formula (I) are of Formula (lie) or (He):
  • the compounds of Formula (I) are of Formula (Ilf):
  • M is -C(0)0- or -OC(O)-
  • M is Ci- 6 alkyl or C2-6 alkenyl
  • R 2 and R 3 are independently selected from the group consisting of C 5 -i 4 alkyl and C 5 -i 4 alkenyl
  • n is selected from 2, 3, and 4.
  • the compounds of Formula (I) are of Formula (lid),
  • each of R 2 and R 3 may be independently selected from the group consisting of C 5 -i 4 alkyl and C 5 -i 4 alkenyl.
  • the compounds of Formula (I) are of Formula (Ilg), (Hg), or their N-oxides, or salts or isomers thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; Mi is a bond or M’; M and M’ are independently selected from -C(0)0-, -OC(O)-, -0C(0)-M”-C(0)0-, -C(0)N(R’)-, -P(0)(0R’)0-, -S-S-, an aryl group, and a heteroaryl group; and R 2 and R 3 are independently selected from the group consisting of H, Ci-i 4 alkyl, and C2-14 alkenyl.
  • M is C1-6 alkyl (e.g., C1-4 alkyl) or C2-6 alkenyl (e.g. C2-4 alkenyl).
  • R2 and R 3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.
  • the ionizable lipids are one or more of the compounds described in U.S. Application Nos. 62/220,091, 62/252,316, 62/253,433, 62/266,460, 62/333,557, 62/382,740, 62/393,940, 62/471,937, 62/471,949, 62/475,140, and 62/475,166, and PCT Application No. PCT/US2016/052352.
  • the ionizable lipids are selected from Compounds 1-280 described in U.S. Application No. 62/475,166.
  • the ionizable lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the ionizable lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the ionizable lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the ionizable lipid is (Compound V), or a salt thereof.
  • a lipid may have a positive or partial positive charge at physiological pH.
  • Such lipids may be referred to as cationic or ionizable (amino)lipids.
  • Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.
  • the ionizable lipids of the present disclosure may be one or more of compounds of formula (III),
  • t 1 or 2;
  • Ai and A 2 are each independently selected from CH or N;
  • Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
  • Ri, R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of C 5-2 o alkyl, C 5-2 o alkenyl, -R”MR’, -R*YR”, -YR”, and -R*OR”;
  • Rxi and Rx 2 are each independently H or Ci- 3 alkyl
  • each M is independently selected from the group consisting of -C(0)0-, -OC(O)-,
  • M* is Ci-C 6 alkyl
  • W 1 and W 2 are each independently selected from the group consisting of -O- and -N(R6>;
  • each R 6 is independently selected from the group consisting of H and C1-5 alkyl
  • X 1 , X 2 , and X 3 are independently selected from the group consisting of a bond, -CH 2 -, -(CH 2 ) 2 -, -CHR-, -CHY-, -C(O)-, -C(0)0-, -0C(0)-, -(CH 2 ) n -C(0)-, -C(0)-(CH 2 ) n -,
  • each Y is independently a C 3-6 carbocycle
  • each R* is independently selected from the group consisting of Ci-i 2 alkyl and C 2-i2 alkenyl;
  • each R is independently selected from the group consisting of Ci- 3 alkyl and a C3-6 carbocycle;
  • each R’ is independently selected from the group consisting of Ci-i 2 alkyl, C 2-i2 alkenyl, and H;
  • each R is independently selected from the group consisting of C 3-12 alkyl, C3 - 1 2 alkenyl and -R*MR’;
  • n is an integer from 1-6;
  • At least one of X 1 , X 2 , and X 3 is not -CH 2 -; and/or
  • Ri, R 2 , R3, R4, and R5 is -R”MR ⁇
  • the compound is of any of formulae (IIIal)-(IIIa8):
  • the ionizable lipids are one or more of the compounds described in U.S. Application Nos. 62/271, 146, 62/338,474, 62/413,345, and 62/519,826, and PCT Application No. PCT/US2016/068300. In some embodiments, the ionizable lipids are selected from Compounds 1-156 described in U.S. Application No. 62/519,826.
  • the ionizable lipids are selected from Compounds 1-16, 42-66, 68-76, and 78-156 described in U.S. Application No. 62/519,826.
  • the ionizable lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the ionizable lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the central amine moiety of a lipid according to Formula (III), (Illal), (IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), or (IIIa8) may be protonated at a physiological pH.
  • a lipid may have a positive or partial positive charge at physiological pH.
  • Such lipids may be referred to as cationic or ionizable (amino)lipids.
  • Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.
  • the lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof.
  • phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and
  • Particular phospholipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • elements e.g., a therapeutic agent
  • Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • an alkyne group can undergo a copper- catalyzed cycloaddition upon exposure to an azide.
  • Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,
  • Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • a phospholipid of the invention comprises l,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1 ,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di- O-octa
  • cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine OChemsPC
  • Cl 6 Lyso PC l-hexadecyl-sn- glycero-3 -phosphocholine
  • 1, 2-dilinolenoyl-sn-glycero-3 -phosphocholine 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE)
  • 1,2- distearoyl-sn-glycero-3-phosphoethanolamine 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine
  • l,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine 1,2- diarachi
  • a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC. In certain embodiments, a phospholipid useful or potentially useful in the present invention is a compound of Formula (IV):
  • each R 1 is independently optionally substituted alkyl; or optionally two R 1 are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R 1 are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl;
  • n 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • A is of the formula
  • each instance of L 2 is independently a bond or optionally substituted Ci -6 alkylene, wherein one methylene unit of the optionally substituted Ci -6 alkylene is optionally replaced with O, N(R n ), S, C(0), C(0)N(R n ), NR N C(0), C(0)0, 0C(0), 0C(0)0, 0C(0)N(R n ), - NR N C(0)0, or NR N C(0)N(R N );
  • each instance of R N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group
  • Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
  • p 1 or 2;
  • R 2 is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.
  • the phospholipids may be one or more of the phospholipids described in U.S. Application No. 62/520,530.
  • a phospholipid useful or potentially useful in the present invention comprises a modified phospholipid head (e.g ., a modified choline group).
  • a phospholipid with a modified head is DSPC, or analog thereof, with a modified quaternary amine.
  • at least one of R 1 is not methyl.
  • at least one of R 1 is not hydrogen or methyl.
  • the compound of Formula (IV) is of one of the following formulae:
  • each v is independently 1, 2, or 3.
  • a compound of Formula (IV) is of Formula (IV-a):
  • a phospholipid useful or potentially useful in the present invention comprises a cyclic moiety in place of the glyceride moiety.
  • a phospholipid useful in the present invention is DSPC, or analog thereof, with a cyclic moiety in place of the glyceride moiety.
  • the compound of Formula (IV) is of Formula (IV-b):
  • a phospholipid useful or potentially useful in the present invention comprises a modified tail.
  • a phospholipid useful or potentially useful in the present invention is DSPC, or analog thereof, with a modified tail.
  • a“modified tail” may be a tail with shorter or longer aliphatic chains, aliphatic chains with branching introduced, aliphatic chains with substituents introduced, aliphatic chains wherein one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof.
  • the compound of (IV) is of Formula (IV-a), or a salt thereof, wherein at least one instance of R 2 is each instance of R 2 is optionally substituted Ci- 30 alkyl, wherein one or more methylene units of R 2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroaryl ene, N(R n ), C(S), C(S)N(R n ), NR N C(S), NR N C(S)N(R n ), S(0), 0S(0), S(0)0, 0S(0)0, 0S(0): 2, -
  • the compound of Formula (IV) is of Formula (IV-c):
  • each x is independently an integer between 0-30, inclusive.
  • G is independently selected from the group consisting of optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroaryl ene, N(R n ), O, S, C(0), C(0)N(R n ), NR N C(0), -
  • Each possibility represents a separate embodiment of the present invention.
  • a phospholipid useful or potentially useful in the present invention comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g, n is not 2). Therefore, in certain embodiments, a phospholipid useful or potentially useful in the present invention is a compound of Formula (IV), wherein n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, a compound of Formula (IV) is of one of the following formulae:
  • a phospholipid useful or potentially useful in the present invention comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful.
  • an alternative lipid is used in place of a phospholipid of the present disclosure.
  • an alternative lipid of the invention is oleic acid.
  • the alternative lipid is one of the following:
  • the lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids.
  • structural lipid refers to sterols and also to lipids containing sterol moieties.
  • Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • sterols are a subgroup of steroids consisting of steroid alcohols.
  • the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol.
  • the structural lipids may be one or more of the structural lipids described in U.S. Application No. 62 /520,530.
  • the lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid.
  • PEG polyethylene glycol
  • PEG-lipid refers to polyethylene glycol (PEG)-modified lipids.
  • PEG-lipids include PEG-modified
  • lipids are also referred to as PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-lipid includes, but not limited to l,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA).
  • PEG-DMG l,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol
  • PEG-DSPE l,2-
  • the PEG-lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the lipid moiety of the PEG-lipids includes those having lengths of from about Ci 4 to about C22, preferably from about Ci 4 to about Ci 6.
  • a PEG moiety for example an mPEG-NEh, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
  • the PEG-lipid is PEG2 k -DMG.
  • the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG.
  • PEG lipid which is a non-diffusible PEG.
  • non-diffusible PEGs include PEG-DSG and PEG-DSPE.
  • PEG-lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.
  • the lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids.
  • a PEG lipid is a lipid modified with polyethylene glycol.
  • a PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • PEG-modified lipids are a modified form of PEG DMG.
  • PEG-DMG has the following structure:
  • PEG lipids useful in the present invention can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain.
  • the PEG lipid is a PEG-OH lipid.
  • a“PEG-OH lipid” (also referred to herein as“hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid.
  • the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain.
  • a PEG-OH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain.
  • a PEG lipid useful in the present invention is a compound of Formula (V).
  • a PEG lipid useful in the present invention is a compound of Formula (V).
  • R 3 is -OR 0 ;
  • R° is hydrogen, optionally substituted alkyl, or an oxygen protecting group
  • r is an integer between 1 and 100, inclusive;
  • L 1 is optionally substituted Ci-io alkylene, wherein at least one methylene of the optionally substituted Ci-io alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroaryl ene, O, N(R n ), S, C(0), C(0)N(R n ), NR N C(0), C(0)0, -
  • D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions:
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
  • A is of the formula
  • each instance of L 2 is independently a bond or optionally substituted Ci -6 alkylene, wherein one methylene unit of the optionally substituted Ci- 6 alkylene is optionally replaced with O, N(R n ), S, C(0), C(0)N(R n ), NR N C(0), C(0)0, OC(0), 0C(0)0, OC(0)N(R n ), -
  • each instance of R 2 is independently optionally substituted Ci- 30 alkyl, optionally substituted Ci- 30 alkenyl, or optionally substituted Ci- 30 alkynyl; optionally wherein one or more methylene units of R 2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroaryl ene, N(R n ), O, S, C(0), C(0)N(R n ), NR N C(0), - NR N C(0)N(R n ), C(0)0, 0C(0), 0C(0)0, 0C(0)N(R n ), NR N C(0)0, C(0)S, SC(0), -
  • each instance of R N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group
  • Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
  • p 1 or 2.
  • the compound of Fomula (V) is a PEG-OH lipid (i.e., R 3 is - OR 0 , and R° is hydrogen).
  • the compound of Formula (V) is of Formula (V-OH): (V-OH),
  • a PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (VI). Provided herein are compounds of Formula (VI): (VI),
  • R 3 is-OR°
  • R° is hydrogen, optionally substituted alkyl or an oxygen protecting group
  • the compound of Formula (VI) is of Formula (VI-OH): (VI-OH),
  • r is 45.
  • the compound of Formula (VI) is:
  • the compound of Formula (VI) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.
  • the PEG-lipids may be one or more of the PEG lipids described in ET.S. Application No. 62/520,530.
  • a PEG lipid of the invention comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified
  • the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG-DPG.
  • a LNP of the invention comprises an ionizable cationic lipid of any of Formula I, II or III, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.
  • a LNP of the invention comprises an ionizable cationic lipid of any of Formula I, II or III, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid comprising a compound having Formula IV, a structural lipid, and the PEG lipid comprising a compound having Formula V or VI.
  • a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid comprising a compound having Formula IV, a structural lipid, and the PEG lipid comprising a compound having Formula V or VI.
  • a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid having Formula IV, a structural lipid, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the invention comprises an ionizable cationic lipid of
  • a LNP of the invention comprises an ionizable cationic lipid of
  • a LNP of the invention comprises an ionizable cationic lipid of
  • an alternative lipid comprising oleic acid, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the invention comprises an ionizable cationic lipid of
  • a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the invention comprises an ionizable cationic lipid of
  • a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VII.
  • a LNP of the invention comprises an N:P ratio of from about 2: 1 to about 30: 1.
  • a LNP of the invention comprises an N:P ratio of about 6: 1.
  • a LNP of the invention comprises an N:P ratio of about 3: 1.
  • a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of from about 10: 1 to about 100: 1.
  • a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 20: 1.
  • a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 10: 1.
  • a LNP of the invention has a mean diameter from about 50nm to about l50nm.
  • a LNP of the invention has a mean diameter from about 70nm to about l20nm.
  • alkyl As used herein, the term “alkyl”, “alkyl group”, or “alkyl ene” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted.
  • C1-14 alkyl means an optionally substituted linear or branched, saturated hydrocarbon including 1-14 carbon atoms. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups.
  • alkenyl means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted.
  • C2-14 alkenyl means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond.
  • An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds.
  • C18 alkenyl may include one or more double bonds.
  • a C18 alkenyl group including two double bonds may be a linoleyl group.
  • an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.
  • alkynyl means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted.
  • C2-14 alkynyl means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon triple bond.
  • An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds.
  • Cl 8 alkynyl may include one or more carbon-carbon triple bonds.
  • an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.
  • the term "carbocycle” or “carbocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings.
  • the notation "C3-6 carbocycle” means a carbocycle including a single ring having 3-6 carbon atoms.
  • Carbocycles may include one or more carbon-carbon double or triple bonds and may be non aromatic or aromatic (e.g., cycloalkyl or aryl groups).
  • Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2 dihydronaphthyl groups.
  • cycloalkyl as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond.
  • carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles.
  • heterocycle or “heterocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom.
  • Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen membered rings.
  • Heterocycles may include one or more double or triple bonds and may be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl groups).
  • heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups.
  • heterocycloalkyl as used herein means a non-aromatic heterocycle and may or may not include any double or triple bond. Unless otherwise specified, heterocycles described herein refers to both unsubstituted and substituted heterocycle groups, i.e., optionally substituted heterocycles.
  • heteroalkyl refers respectively to an alkyl, alkenyl, alkynyl group, as defined herein, which further comprises one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus) wherein the one or more heteroatoms is inserted between adjacent carbon atoms within the parent carbon chain and/or one or more heteroatoms is inserted between a carbon atom and the parent molecule, i.e., between the point of attachment.
  • heteroatoms e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus
  • heteroalkyls, heteroalkenyls, or heteroalkynyl s described herein refers to both unsubstituted and substituted heteroalkyls, heteroalkenyls, or heteroalkynyl s, i.e., optionally substituted heteroalkyls, heteroalkenyls, or heteroalkynyl s.
  • a “biodegradable group” is a group that may facilitate faster metabolism of a lipid in a mammalian entity.
  • a biodegradable group may be selected from the group consisting of, but is not limited to, -C(0)0-, -OC(O)-, -C(0)N(R')-, -N(R')C(0)-, -C(0)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(0)(0R)0-, -S(0)2-, an aryl group, and a heteroaryl group.
  • an "aryl group” is an optionally substituted carbocyclic group including one or more aromatic rings.
  • aryl groups include phenyl and naphthyl groups.
  • a "heteroaryl group” is an optionally substituted heterocyclic group including one or more aromatic rings.
  • heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted.
  • M and M' can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole.
  • M and M' can be independently selected from the list of biodegradable groups above.
  • aryl or heteroaryl groups described herein refers to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.
  • Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified.
  • R is an alkyl or alkenyl group, as defined herein.
  • the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein.
  • a Cl -6 alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.
  • N-oxides can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to afford other compounds of the disclosure.
  • an oxidizing agent e.g., 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides
  • mCPBA 3-chloroperoxybenzoic acid
  • hydrogen peroxides hydrogen peroxides
  • all shown and claimed nitrogen-containing compounds are considered, when allowed by valency and structure, to include both the compound as shown and its N-oxide derivative (which can be designated as N->0 or N + -0 ).
  • the nitrogens in the compounds of the disclosure can be converted to N-hydroxy or N-alkoxy compounds.
  • N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as m CPBA.
  • nitrogen-containing compounds are also considered, when allowed by valency and structure, to cover both the compound as shown and its N-hydroxy (i.e., N-OH) and N-alkoxy (i.e., N-OR, wherein R is substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3-l4-membered carbocycle or 3- l4-membered heterocycle) derivatives.
  • N-OH N-hydroxy
  • N-alkoxy i.e., N-OR, wherein R is substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3-l4-membered carbocycle or 3- l4-membered heterocycle
  • the lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above.
  • the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components.
  • a permeability enhancer molecule can be a molecule described by U.S. Patent Application Publication No.2005/0222064.
  • Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).
  • a polymer can be included in and/or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form).
  • a polymer can be biodegradable and/or biocompatible.
  • a polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • the ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt/wt).
  • the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1,21:1,22:1,23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1,41:1,42:1,43:1,44:1,45:1,46:1,47:1,48:1,49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt). In some embodiments, the wt/wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.
  • the pharmaceutical composition disclosed herein can contain more than one polypeptides.
  • a pharmaceutical composition disclosed herein can contain two or more polynucleotides (e.g., RNA, e.g., mRNA).
  • the lipid nanoparticles described herein can comprise
  • polynucleotides e.g., mRNA
  • lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about 20: 1, from about 5: 1 to about 25: 1, from about 5: 1 to about 30:1, from about 5: 1 to about 35:1, from about 5: 1 to about 40: 1, from about 5: 1 to about 45: 1, from about 5: 1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about
  • the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.
  • the pharmaceutical compositions disclosed herein are formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) a polynucleotide encoding a ABCB4,
  • nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) a polynucleotide encoding an ABCB4 polypeptide, a polynucleotide encoding an ABCB11 polypeptide, and a polynucleotide encoding an
  • the lipid composition disclosed herein can encapsulate the polynucleotide(s) encoding an ABCB4, ABCB11, or
  • Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer.
  • Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.
  • LNPs lipid nanoparticles
  • liposomes e.g., lipid vesicles
  • lipoplexes e.g., lipoplexes.
  • a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.
  • Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes.
  • LNPs lipid nanoparticles
  • nanoparticle compositions are vesicles including one or more lipid bilayers.
  • a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments.
  • Lipid bilayers can be functionalized and/or crosslinked to one another.
  • Lipid bilayers can include one or more ligands, proteins, or channels.
  • a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and mRNA.
  • the LNP comprises an ionizable lipid, a PEG-modified lipid, a sterol and a structural lipid.
  • the LNP has a molar ratio of about 20-60% ionizable lipid: about 5-25% structural lipid: about 25- 55% sterol; and about 0.5-15% PEG-modified lipid.
  • the LNP has a polydispersity value of less than 0.4. In some embodiments, the LNP has a net neutral charge at a neutral pH. In some embodiments, the LNP has a mean diameter of 50-150 nm. In some embodiments, the LNP has a mean diameter of 80-100 nm.
  • lipid refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic.
  • lipids examples include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids.
  • the amphiphilic properties of some lipids leads them to form liposomes, vesicles, or membranes in aqueous media.
  • a lipid nanoparticle may comprise an ionizable lipid.
  • an ionizable lipid has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties.
  • an ionizable lipid may be positively charged or negatively charged.
  • An ionizable lipid may be positively charged, in which case it can be referred to as“cationic lipid”.
  • an ionizable lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipid.
  • a“charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc.

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Abstract

La présente invention concerne des compositions d'acides nucléiques se rapportant à des transporteurs épithéliaux biliaires. Par exemple, la présente invention concerne des acides nucléiques capables de réguler la sécrétion biliaire de phospholipides, y compris la phosphatidylcholine, par exemple celles codées par le membre 4 de la sous-famille B de la cassette de liaison à l'ATP (ABCB4) ou un fragment biologiquement actif de celles-ci, dans une cellule cible. Dans un mode de réalisation préféré, la présente invention concerne des compositions comprenant un ARNM modifié codant l'ABCB4 formulé dans un support de nanoparticules lipidiques (LNP) et des constructions dérivées, qui sont utiles pour traiter ou prévenir le type 3 de cholestase intrahépatique familiale progressive (PFIC3).
PCT/US2019/051172 2018-09-13 2019-09-13 Arnm modifié pour le traitement de troubles de la cholestase intrahépatique familiale progressive WO2020056370A1 (fr)

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JP2021514132A JP2022500443A (ja) 2018-09-13 2019-09-13 進行性家族性肝内胆汁うっ滞障害を処置するための修飾ｍＲＮＡ
AU2019338557A AU2019338557A1 (en) 2018-09-13 2019-09-13 Modified mRNA for the treatment of progressive familial intrahepatic cholestasis disorders
CA3111836A CA3111836A1 (fr) 2018-09-13 2019-09-13 Arnm modifie pour le traitement de troubles de la cholestase intrahepatique familiale progressive
EP19861226.9A EP3863645A4 (fr) 2018-09-13 2019-09-13 Arnm modifié pour le traitement de troubles de la cholestase intrahépatique familiale progressive
US17/276,112 US20220054653A1 (en) 2018-09-13 2019-09-13 Modified mrna for the treatment of progressive familial intrahepatic cholestasis disorders

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